Exendin analogues, processes for their preparation and medicaments containing them

ABSTRACT

The invention concerns exendin analogues which can be used in the treatment of diabetes mellitus. The invention also concerns processes for preparing these substances and medicaments containing them. The exendin analogues are derived from SEQ ID:1(I) or SEQ ID:2(II).

This application is a 371 of PCT/EP97/02930 filed Jun. 5, 1997 claimingpriority to DE 196 22 502.7 filed Jun. 5, 1996 and DE 196 37 230.5 filedSep. 13, 1996.

This invention concerns new exendin analogues which can be used in thetherapy of diabetes mellitus, processes for their production andpharmaceutical preparations containing them.

BACKGROUND OF THE INVENTION

A functional connection between the small intestine and exocrinepancreas was proven in the 1960's after it became possible to accuratelydetermine insulin in plasma. The insulin response after oral glucoseadministration is much stronger than after intravenous glucoseadministration even if identical plasma levels of glucose are reached.This “incretin effect” is explained by the functional combination of theentero-insular axis. Intestinal hormones are responsible for this effectwhich are released from the small intestine after meals, circulate inthe plasma at increased measurable levels and amplify glucose-inducedinsulin release. In addition to the classical incretin hormone gastricinhibitory polypeptide I (GIP), glucagon-like peptide. 1 (GLP-1) isnowadays of primary interest. In a relatively short time GLP-1 hasmatured from being the physiologically most interesting incretin hormonecandidate to a potential alternative for the treatment of diabetesmellitus type II. The present invention describes new substances whichimitate the effect of the naturally occurring GLP-1 molecule. These newsubstances are characterized by an increased stability while maintainingefficacy.

Anti-diabetogenic Action

Infusion and subcutaneous injection of GLP-1 cause a considerableincrease of insulin secretion and an inhibition of glucagon release inpatients with diabetes mellitus type II (Gutniak, M. (1992); Kreymann,B. (1987); Nathan, D. M. (1992); Nauck, M. A. (1993a & b)). Both are oftherapeutic interest and are involved in the blood sugar lowering effectof GLP-1: insulin promotes glucose uptake by its target tissue andinhibits gluconeogenesis. Furthermore GLP-1 analogues would be expectedto increase glucose uptake in the periphery. The inhibition of glucagonsecretion must be regarded as an indirect GLP-1 effect sinceglucagon-producing A cells express no GLP-1 receptors (Komatsu, R.(1989)). On the contrary, the increased insulin and somatostatin releaseappear to be the decisive factor. Both hormones are known as inhibitorsof glucagon release.

Two molecular mechanisms certainly contribute to the GLP-1-inducedinsulin release in diabetes mellitus type II. In addition to directlyamplifying the glucose-induced insulin release, GLP-1 sensitizes asubgroup of B cells towards the key stimulus “glucose” (Fehmann, H. C.(1991)) and possibly also towards further stimuli so that overall more Bcells secrete insulin. This prizing affect is the most likelyexplanation for the fact that GLP-1 leads to a prolonged release ofinsulin despite its relatively short plasma half-life.

This effect depends on increased glucose levels (>108 mg/dl) (Göke, R.(1993a)). It distinguishes GLP-1 fundamentally from the sulfonylureaswhich influence insulin secretion independently of the plasma level ofglucose. If the glucose value decreases below 108 mg/dl, the insulinsecretion dries up even with an intravenous infusion of GLP-1. Hencehypoglycaemias would be hardly expected when GLP-1 is usedtherapeutically. In fact they have also not been described in theprevious clinical studies. However, the pharmacokinetic properties ofGLP-1 are problematic. The duration of action is limited due to its veryshort half-life.

From a therapeutic point of view the synthesis of stable and stronglyeffective GLP-1 peptide analogues is in any case desirable. Peptideanalogues have now been synthesized based on the molecule exendin thatwas originally isolated from the venom of lizards with the aim ofdeveloping improved therapeutic agents that are stable towardsdegradation with an increased duration of action for the treatment ofdiabetes mellitus. These peptides have the same pharmacological effectas GLP-1, but surprisingly have a considerably longer half-life.

The new peptide sequences described as the subject matter of theinvention have an effect on insulin synthesis and insulin release and anaction on the insulin effect especially the uptake of glucose into thetarget tissues, muscle and fat tissue as well as emptying of thestomach.

SUBJECT MATTER OF THE INVENTION

The present invention is based on the sequence of exendin-3 andexendin-4, peptides which were isolated from the secretory product ofHeloderma horridum or Heloderma suspectum (Eng. J. et al. (1990, 1992)).The amino acid sequence and effect of the two peptides on the pancreashas already been published by several authors (Eng. J. et a. (1990);Raufman, J. P. (1992); Göke, R. (1993b); Thorens, B. (1993)). Thesubject matter of this invention are new truncated exendin fragmentswhich comprise the amino acid sequences of exendin-3-(1-30) orexendin-4-(1-30) in which the C-terminal end of these sequences can beshortened by up to 3 amino acids, preferably by at most 1 amino acid,and the N-terminal end can be shortened by up to 2 and preferably atmost 1 amino acid. Surprisingly these exendin fragments are biologicallyactive although the amino acid sequence is shortened. Shortened aminoacid sequences are more economical to produce than relatively longersequences. Hence, peptide fragments with the following sequences areparticularly preferred; especially peptide fragments that are based onexendin-3-(1-30) (SEQ ID NO.1):

SEQ ID NO:1 based on exendin-3

1       5         10        15 H S D G T F T S D L S K Q M E E E A V20        25        30 R L F I E W L K N G X₁

SEQ ID NO:2 based on exendin-4

1       5         10        15 H G E G T F T S D L S K Q M E E E A V20        25        30 R L F I E W L K N G X₁

in which the amino acids at position 1, 2, 28, 29 or 30 can be part ofthe sequence depending on the desired chain length. The peptides arenumbered through from the N-terminus to the C-terminus. X₁ denotes aproteogenic or non-proteogenic amino acid apart from glycine. Exendinand exendin analogues with a chain length of 1-27 are preferred andespecially those with a chain length of 1-30.

The carboxyl group COR₃ of the amino acid at the C-terminal end can bepresent in a free form (R₃=OH) or in the form of a physiologicallytolerated alkaline or alkaline earth salt such as e.g. a sodium,potassium or calcium salt. The carboxyl group can also be esterifiedwith primary, secondary or tertiary alcohols such as e.g. methanol,branched or unbranched C₁-C₆-alkyl alcohols, in particular ethyl alcoholor tert. butanol. The carboxyl group can, however, also be amidated withprimary or secondary amines such as ammonia, branched or unbranchedC₁-C₆ alkylamines or C₁-C₆ di-alkylamines, in particular methylamine ordimethylamine.

The amino group of the amino acid NR₁R₂ at the N-terminal end can bepresent in a free form (R₁, R₂=H) or in the form of a physiologicallytolerated salt such as e.g. a chloride or acetate. The amino group canalso be acetylated with acids so that R₁=H and R₂=acetyl,trifluoroacetyl, adamantyl or be present in a form protected byconventional amino protecting groups of peptide chemistry such as e.g.Fmoc, Z, Boc, Alloc or be N-alkylated in which R₁ and/or R₂=C₁-C₆ alkylor C₂-C₈ alkenyl or C₇-C₉ aralkyl.

Alkyl residues are understood as straight-chained, branched oroptionally ring-shaped alkyl residues, preferably methyl, ethyl,isopropyl and cyclohexyl.

All exendin fragments can be present as completely or partiallyprotected derivatives.

A further subject matter of this invention are exendin fragments withthe above-mentioned properties and sequence lengths in which at leastone but at most eleven of the modifications listed under (a) to (p) havebeen additionally carried out. Exendin fragments are preferred whichhave at most nine and particularly preferably those which have at mostfive of the modifications listed under (a) to (p).

(a) The α-amino acid in position 1 is D-His, Ala, D-Ala, Gly, Lys orD-Lys of which Ala, Gly or Lys are particularly preferred; or

(b) the α-amino acid in position 2 is Ser, D-Ser, Thr, D-Thr, Gly, Ala,D-Ala, Ile, D-Ile, Val, D-Val, Leu or D-Leu, preferably Ser, Thr, Gly,Ala, Val, Ile or Leu; or

(c) the α-amino acid in position 3 is Glu, D-Glu, Asp, D-Asp, Ala orD-Ala, preferably Glu, Asp or Ala; or

(d) the amino acid in position 4 is Ala, D-Ala or β-Ala, preferably Ala;or

(e) the α-amino acid in position 5 is Ser, Tyr or Ala; or

(f) the α-amino acid in position 6 is Ala, Ile, Val, Leu, Cha or Tyr,preferably Ala, Ile, Val, Leu or Tyr; or

(g) the α-amino acid in position 7 is Ala, D-Ala, Tyr, D-Tyr, Ser, D-Seror D-Thr, preferably Ala, Tyr or Ser; or

(h) the α-amino acid in position 8 is Ala, Tyr or Thr; or

(i) the α-amino acid in position 9 is Ala, D-Ala, Glu, D-Glu or D-Asp,preferably Ala or Glu; or

(j) the amino acids in position 10, 11, 12, 15, 16, 17, 18, 19, 20, 21,24, 28, 29 are independently of one another a proteinogenic ornon-proteinogenic D- or L-amino acid, preferably a proteinogenic L-aminoacid; or

(k) the α-amino acid in position 13 is a neutral L-amino acid,preferably a neutral proteinogenic L-amino acid; or

(l) the α-amino acid in position 14 is, for the purposes ofstabilization, replaced by a neutral L- or D-amino acid, apart fromL-leucine, preferably by Nle, D-Nle, Ala, D-Ala, Ile, D-Ile, Val orD-Val, wherein Ile, Val or Ala are particularly preferred; or

(m) the α-amino acid in position 22 is D-Phe, Tyr, D-Tyr, Leu, D-Leu,Val, D-Val, L-Cha, D-Cha, β-1-Nal, β-2-Nal or β-1-D-Nal, wherein Tyr,Leu or Val are preferred; or

(n) the α-amino acid in position 23 is Leu, D-Leu, D-Ile, Val, D-Val,L-Cha, D-Cha, Tyr, D-Tyr, Phe or D-Phe, wherein Leu, Val, Tyr or Phe arepreferred; or

(o) the α-amino acid in position 25, 26 or 27 is a neutral L- or D-aminoacid, preferably a neutral, proteinogenic L-amino acid; or

(p) the α-amino acid in position 30 is a proteinogenic ornon-proteinogenic D- or L-amino acid apart from glycine, preferably Arg,D-Arg, Tyr or D-Tyr, Arg or Tyr are particularly preferred.

Among the new exendin fragments, those are particularly preferred whichcontain the amino acid leucine at position 10 and/or the amino acidvaline at position 19, the amino acid isoleucine or alanine instead ofmethionine at position 14 and arginine at position 30 in addition to thealready mentioned properties and sequence lengths. Those modificationsof exendin fragments are also particularly preferred in which, inaddition to the particularly preferred amino acids at positions 10, 14,19 and 30, one of the 20 known proteinogenic L-amino acids is located atposition 2.

Preferred exendin analogues have a substitution at position 3 or 14,particularly preferably at position 2 and especially preferably theexendin analogues only contain proteinogenic amino acids.

In addition to new shortened and stabilized exendin-3 and exendin-4analogues, the invention also concerns processes for producing theseanalogues in which the analogues are prepared in a solid phase synthesisfrom protected amino acids contained in the analogues which are coupledin sequence and correspond to the amino acids in the analogues and whichare optionally supplemented with corresponding amino acids which do notoccur in the natural exendin peptides.

The glycine at position 30 of the exendin-3 or exendin-4 sequence wassubstituted by another proteogenic or non-proteogenic amino acid inorder to avoid diketopiperazine formation during the synthesis aftercleavage of the amino terminal protective group.

The exendin-(1-30) analogues and fragments are advantageous compared tothe exendins-1-(1-39) since the shorter sequences of these analoguesenable a more simple synthesis in higher yields.

The abbreviations and definitions of the amino acids that are used wererecommended in Pure Appl. Chem. 31, 639-45 (1972) and ibid. 40, 277-90(1974) and correspond to the PCT rules (WIPO standard st.23:Recommendation for the Presentation of Nucleotide and Amino AcidSequences in Patent Applications and in Published Patent Documents). Theone and three letter codes are as follows:

Amino acid abbreviations three one Amino acid letter code letter codealanine Ala A arginine Arg R asparagine Asn N aspartic acid Asp Dcysteine Cys C glutamine Gln Q glutamic acid Glu E glycine Gly Ghistidine His H isoleucine Ile I leucine Leu L lysine Lys K methionineMet M phenylalanine Phe F proline Pro P serine Ser S threonine Thr Ttryptophan Trp W tyrosine Tyr Y valine Val V other amino acids Xaa X

The abbreviations represent L-amino acids if not specified otherwisesuch as D- or D,L-. The D-amino acids are written in small letters inthe one letter code. Certain natural as well as non-natural amino acidsare achiral e.g. glycine. In the representation the N-terminal end ofall peptides is on the left and the C-terminal end is on the right.

Examples of non-proteinogenic amino acids are given in the followinglist together with their abbreviations:

β-alanine β-Ala o-aminobenzoic acid Oab m-aminobenzoic acid Mabp-aminobenzoic acid Pab m-aminomethylbenzoic acid Amb ω-aminohexanoicacid Ahx ω-aminoheptanoic acid Ahp ω-aminooctanoic acid Aocω-aminodecanoic acid Ade ω-aminotetradecanoic acid Atd citrulline Citcyclohexylalanine Cha α,γ-diaminobutyric acid Dab α,β-diaminopropionicacid Dap methionine sulfoxide Met(O) c^(α)-methyl-alanine AibN-methyl-glycine (sarcosine) Sar naphthylalanine Nal norleucine Nleornithine Orn 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid Tic

All amino acids can be divided into the following three main classesaccording to their physical-chemical properties:

Acidic: The amino acid releases a proton in aqueous solution and atphysiological pH and consequently carries a negative charge.

Basic: The amino acid accepts a proton in aqueous solution and atphysiological pH and consequently carries a positive charge.

Neutral: The amino acid is in an uncharged state in aqueous solution andat physiological pH.

The definition “carries a positive/negative charge” or “is in anuncharged state” only applies when on statistical average a significantnumber of a class of amino acids (at least 25%) are in the state.

In addition to the 20 so-called proteinogenic amino acids whoseincorporation into proteins is controlled by the information of thegenetic code, non-proteinogenic amino acids can also be incorporatedinto peptide sequences by the described synthesis process. A list of theproteinogenic amino acids and their classification into theabove-mentioned three classes is given in Table 1. Non-proteinogenicamino acids are not genetically coded. Examples of non-proteinogenicamino acids and their classification into acidic, basic or neutral aminoacids is given in Table 1.

TABLE 1 proteinogenic non-proteinogenic acidic Asp, Glu basic Arg, His,Lys Dab, Dap, Orn neutral Ala, Asn, Cys, Gln, Gly, β-Ala, Aib, Cit, Cha,Ile, Leu, Met, Phe, Pro, Oab, Mab, Pab, Amb, Ahx, Ser, Thr, Trp, Tyr,Val Ahp, Aoc, Ade, Atd, Nal, Nle, Sar, Tic

The exendin analogoues which are a subject matter of the invention haveadvantageous therapeutic properties. Hence they lead to a stimulation ofinsulin release from the endocrine pancreas, to an increase of insulinbiosynthesis and to increased peripheral glucose utilization. Sincethese effects can only be observed when the blood sugar levels are atthe same time increased, a hypoglycemia would not be expected to occurafter their administration. Furthermore the exendin analogues inhibitglucagon release from the endocrine pancreas and lead to a decrease ofgluconeogenesis. In non-insulin dependent diabetes mellitus (NIDDM) theexendin analogues result in a considerable improvement of the metabolicsituation. In particular the glucose uptake in muscle and fat tissue isincreased independently of the insulin secretory effect. Due to theinhibitory effect on glucagon release, it is also appropriate toadminister the exendin analogues in insulin dependent diabetes mellitus.Compared to glucagon-like peptide 1 (GLP-1) and the known exendin-3 andexendin-4 sequences, the exendin analogues according to the inventionsurprisingly have a higher efficacy in the various test systems so thatthey are more suitable for a therapeutic application than GLP-1,exendin-3 or exendin-4. The advantages of the new exendin analogues arein particular as follows: higher stability towards degradation andmetabolism, longer duration of action, effectiveness at lower doses.Analogues based on exendin-3 are particularly preferred which exhibitparticularly long durations of action or effectiveness at particularlylow doses.

Solid phase and liquid phase synthesis is a conventional process forsynthesizing peptides. In order to optimize the process for thesynthesis of a particular product with regard to the purity of the crudeproduct and yield, it is necessary that the process parameters and thematerials that are used, for example the support material, the reagentswhich should make groups react, the materials for blocking the groupswhich should not react or the reagents which cleave blocking materialsare adapted to the product to be synthesized, to the intermediateproducts to be synthesized and the starting materials. This adaptationis not simple with regard to the interdependency of the many processparameters.

Pharmaceutical preparations which contain the peptides according to theinvention individually or together as an active substance in addition toconventional auxiliary substances and additives are preferablyadministered parenterally (subcutaneously, intramuscularly orintravenously). However, all other common forms of administration suchas oral, rectal, buccal (including sublingual), pulmonary, transdermal,iontophoratic, vaginal and intranasal administration come intoconsideration. The drug has an insulin-regulating effect therebypromoting in an advantageous manner the compensation of the blood sugarlevel. It is advantageous for the use of the drug when blood levelsbetween 20 and 50 pmol/l are attained. Infusion rates of 0.4-1.2pmol/kg/min are necessary for this. In the case of a subcutaneous orbuccal administration, substance quantities of 5-500 nmol are necessarydepending on the galenic form and intended duration of action.

The exendin analogues according to the invention or pharmacologicallyacceptable salts thereof are preferably stored as sterile lyophilisatesand mixed with a suitable isotonic solution before administration. Theanalogues can then be injected, infused or optionally also absorbedthrough the mucous membranes in this form. The conventional isotonicaqueous systems that are suitable for injection or infusion whichcontain common additives for injection solutions such as stabilizers andsolubilizers can be used as solvents. Physiological saline solution oroptionally solutions made isotonic by buffers are preferred in thiscase.

Additives are, for example, tartrate or citrate buffer, ethanol,complexing agents (such as ethylene diamintetraacetic acid and non-toxicsalts thereof), high molecular polymers (such as liquid polyethyleneoxide) to regulate the viscosity. Liquid carrier substances forinjection solutions must be sterile and are preferably filled intoampoules. Solid carrier substances are for example starch, lactose,mannitol, methyl cellulose, talcum, highly dispersed silicic acids,higher molecular weight fatty acids (such as stearic acid), gelatin,agar-agar, calcium phosphate, magnesium stearate, animal or vegetablefats, solid high molecular polymers (such as polyethylene glycol);suitable preparations for oral administration can if desired containflavourings or sweeteners. For nasal administration surfactants can beadded to improve absorption through the nasal mucous membrane e.g.cholic acid, taurocholic acid, chenodeoxycholic acid, glycolic acid,dehydrocholic acid, deoxycholic acid and cyclodextrins.

The daily dose to be administered is in a range of 150-500 nmol. Thedetermination of the biological activity is based on measurementscompared to international reference preparations for glucagon-likepeptide-1, exendin-3 or exendin-4 in a conventional test procedure forglucagon-like peptide-1.

The exendin analogues according to the invention can be prepared byconventional processes in peptide synthesis as described for example inJ. M. Steward and J. D. Young “Solid Phase Peptide Synthesis”, 2nd ed.,Pierce Chemical Co., Rockford, Ill. (1984) and in J. Meienhofer HormonalProteins and Peptides, Vol.2 Academic Press, New York (1973) for solidphase synthesis and in E. Schroder and K. Lubke “The Peptides”, Vol.1,Academic Press, New York (1965) for liquid phase synthesis.

General Processes for Peptide Synthesis

In general protected amino acids are added to a growing peptide chainfor the synthesis of peptides. Either the amino group or the carboxylgroup as well as any reactive group in the side chain of the first aminoacid are protected. This protected amino acid is either coupled to aninert support or it can also be used in solution. The next amino acid inthe peptide sequence is appropriately protected under conditions whichfavour the formation of an amide bond and is added to the first. Afterall desired amino acids have been coupled in the correct sequence, theprotective groups and optionally the support phase are cleaved. Thecrude polypeptide that is obtained is reprecipitated and preferablypurified chromatographically to form the final product.

A preferred method for synthesizing analogues of physiologically activepolypeptides with fever than fourty amino acids comprises a solid phasepeptide synthesis. In this method the α-amino functions (N^(α)) and anyreactive side chains are protected with acid-labile or base-labilegroups. The protective groups that are used should be stable under theconditions for linking amide bonds but it should be possible to readilycleave them without impairing the polypeptide chain that has formed.Suitable protective groups for the α-amino function include thefollowing groups but are not limited to these: t-butyloxycarbonyl (Boc),benzyloxycarbonyl (Z), o-chlorbenzyloxycarbonyl,bi-phenylisopropyloxycarbonyl, tert.-amyloxycarbonyl (Amoc),α,α-dimethyl-3,5-dimethoxy-benzyloxycarbonyl, o-nitrosulfenyl,2-cyano-t-butoxy-carbonyl, 9-fluorenylmethoxycarbonyl (Fmoc),1-(4,4-dimethyl-2,6-dioxocylohex-1-ylidene)ethyl (Dde) and the like.9-Fluorenylmethoxycarbonyl (Fmoc) is preferably used as theN^(α)-protective group.

Suitable side chain protective groups include the following but are notlimited to these: acetyl, allyl (All), allyloxycarbonyl (Alloc), benzyl(Bzl), benzyloxycarbonyl (Z), t-butyloxycarbonyl (Boc), benzyloxymethyl(Bom), o-bromobenzyloxycarbonyl, t-butyl (tBu), t-butyldimethylsilyl,2-chlorobenzyl, 2-chlorobenzyloxycarbonyl (2-CIZ), 2,6-dichlorobenzyl,cyclohexyl, cyclopentyl,1-(4,4-dimethyl-2,6-dioxocyclohex-1-ylidene)ethyl (Dde), isopropyl,4-methoxy-2,3-6-trimethylbenzylsulfonyl (Mtr),2,3,5,7,8-pentamethylchroman-6-sulfonyl (Pmc), pivalyl,tetrahydropyran-2-yl, tosyl (Tos), 2,4,6-trimethoxybenzyl,trimethylsilyl and trityl (Trt).

In the solid phase synthesis the C-terminal amino acid is coupled as thefirst to a suitable support material. Suitable support materials arethose which are inert towards the reagents and reaction conditions forthe stepwise condensation and cleavage reactions and which do notdissolve in the reaction media that are used. Examples of commerciallyavailable support materials include styrene/divinylbenzene copolymerswhich have been modified with reactive groups and/or polyethylene glycoland also chloromethylated styrene/divinylbenzene copolymers,hydroxymethylated or aminomethylated styrene/divinylbenzene copolymersand the like. Polystyrene (1%)-divinylbenzene or TentaGel® (RappPolymere, Tübingen) derivatized with 4-benzyloxybenzyl-alcohol(Wang-anchor (Wang, S. S. 1973)) or 2-chlorotrityl chloride (Barlos, K.et al. 1989) is preferably used if it is intended to prepare thepeptidic acid. In the case of the peptide amide, polystyrene (1%)divinylbenzene or TentaGel® derivatized with5-(4′-aminomethyl)-3′,5′-dimethoxyphenoxy)valeric acid (PAL-anchor)(Albericio, F. et al. 1987) or p-(2,4-dimethoxyphenyl-aminomethyl)-phenoxy group (Rink-Amid anchor (Rink, H. 1987)) is preferred.

The linkage to the polymeric support can be achieved by reacting theC-terminal Fmoc-protected amino acid with the support material with theaddition of an activation reagent in ethanol, acetonitrile,N,N-dimethylformamide (DMF), dichloromethane, tetrahydrofuran,N-methylpyrrolidone or similar solvents preferably in DMF at roomtemperature or elevated temperatures e.g. between 40° C. and 60° C.,preferably at room temperature and with reaction times of 2 to 72 hours,preferably about 2×2 hours.

The coupling of the N^(α) protected amino acid preferably the Fmoc aminoacid to the PAL, Wang or Rink anchor can for example be carried out withthe aid of coupling reagents such as N,N′-dicyclohexylcarbodiimide(DCC), N,N′-diisopropylcarbodiimide (DIC) or other carbodiimides,2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium tetrafluoroborate(TBTU) or other uronium salts, o-acyl-ureas,benzotriazol-1-yl-tris-pyrrolidino-phosphonium hexafluorophosphate(PyBOP) or other phosphonium salts, N-hydroxysuccinimides, otherN-hydroxyimides or oximes in the presence or also in the absence of1-hydroxybenzotriazole or 1-hydroxy-7-azabenzotriazole preferably withthe aid of TBTU with addition of HOBt, with or without the addition of abase such as for example diisopropylethylamine (DIEA), triethylamine orN-methylmorpholine, preferably diisopropylethylamine with reaction timesof 2 to 72 hours, preferably 3 hours in a 1.5 to 3-fold excess of theamino acid and the coupling reagents, preferably in a 2-fold excess andat temperatures between about 10° C. and 50° C., preferably 25° C. in asolvent such as dimethylformamide, N-methylpyrrolidone ordichloromethane, preferably dimethylformamide. Instead of the couplingreagents it is also possible to use the active esters (e.g.pentafluorophenyl, p-nitrophenyl or the like), the symmetric anhydrideof the N^(α)-Fmoc-amino acid, its acid chloride or acid fluoride underthe conditions described above.

The N^(α)-protected amino acid, preferably the Fmoc amino acid ispreferably coupled to the 2-chlorotrityl resin in dichloromethane withthe addition of DIEA with reaction times of 10 to 120 minutes,preferably 20 minutes but is not limited to the use of this solvent andthis base.

The successive coupling of the protected amino acids can be carried outaccording to conventional methods in peptide synthesis typically in anautomated peptide synthesizer. After cleavage of the N^(α)-Fmocprotective group of the coupled amino acid on the solid phase bytreatment with piperidine (10% to 50%) in dimethylformamide for 5 to 20minutes, preferably 2×2 minutes with 50% piperidine in DMF and 1×15minutes with 20% piperidine in DMF, the next protected amino acid in a 3to 10-fold excess, preferably in a 10-fold excess is coupled to theprevious amino acid in an inert, non-aqueous, polar solvent such asdichloromethane, DMF or mixtures of the two, preferably DMF and attemperatures between about 10° C. and 50° C., preferably at 25° C. Thereagents that have already been mentioned for coupling the firstN^(α)-Fmoc amino acid to the PAL, Wang or Rink anchor are suitable ascoupling reagents. Active esters of the protected amino acid, orchlorides or fluorides or symmetric anhydrides thereof can also be usedas an alternative.

At the end of the solid phase synthesis the peptide is cleaved from thesupport material while simultaneously cleaving the side chain protectinggroups. Cleavage can be carried out with trifluoroacetic acid or otherstrongly acidic media with addition of 5%-20% V/V scavengers such asdimethylsulfide, ethylmethylsulfide, thioanisole, thiocresol, m-cresol,anisole ethanedithiol, phenol or water, preferably 15% v/vdimethylsulfide/ethanedithiol/m-cresol 1:1:1 within 0.5 to 3 hours,preferably 2 hours. Peptides with fully protected side chains areobtained by cleaving the 2-chlorotrityl anchor with glacial aceticacid/trifluoroethanol/dichloromethane 2:2:6. The protected peptide canbe purified by chromatography on silica gel. If the peptide is linked tothe solid phase via the Wang anchor and if it is intended to obtain apeptide with a C-terminal alkylamidation, the cleavage can be carriedout by aminolysis with an alkylamine or fluoroalkylamine. The aminolysisis carried out at temperatures between about −10° C. and 50° C.,preferably about 25° C. and reaction times between about 12 and 24hours, preferably about 18 hours. In addition the peptide can also becleaved from the support by re-esterification e.g. with methanol.

The acidic solution that is obtained is admixed with a 3 to 20-foldamount of cold ether or n-hexane, preferably a 10-fold excess of diethylether, in order to precipitate the peptide and hence to separate thescavengers and cleaved protective groups that remain in the ether. Afurther purification can be carried out by re-procipitating the peptideseveral times from glacial acetic acid. The precipitate that is obtainedis taken up in water or tert. butanol or mixtures of the two solvents,preferably a 1:1 mixture of tert, -butanol/water and freeze-dried.

The peptide obtained can be purified by some or all of the followingchromatographic methods: ion exchange over a weakly basic resin in theacetate form; hydrophobic adsorption chromatography on non-derivatizedpolystyrene/divinylbenzene copolymers (e.g. Amberlite® XAD); adsorptionchromatography on silica gel; ion exchange chromatography e.g. oncarboxymethyl cellulose; distribution chromatography e.g. on Sephadex®G-25; countercurrent distribution chromatography; or high pressureliquid chromatography (HPLC) in particular reversed-phase HPLC on octylor octadecylsilylsilica (ODS) phases.

In summary part of the present invention encompasses processes for thepreparation of polypeptides and pharmaceutically usable salts thereof.These processes which lead to physiologically active shortenedhomologues and analogues of exendin-3 or exendin-4 with theabove-mentioned preferred chain lengths and modifications compriseprocesses for the sequential condensation of protected amino acids on asuitable support material, methods for cleaving the support andprotective groups and for purifying the crude peptides that areobtained.

The amino acid analysis Was carried out with an amino acid analyzer 420A from the Applied Biosystems Company (Weiterstadt). 50 to 1000 pmol ofthe sample to be analysed was applied to the sample carrier in 10 to 40μl solution and subsequently fully automatically hydrolysed for 90minutes in the gas phase at 160° C. with 6 N hydrochloric acid,derivatized with phenylisothiocyanate and analysed on-line by amicrobore HPLC. Mass spectroscopic examinations were carried out on anAPI III triple-quadrupole mass spectrometer (SCIEX; Thornhill, Canada)equipped with an ion spray ion source.

The protected amino acid derivatives can for example be obtained fromNovabiochem GmbH (Bad Soden).

The following examples only represent an illustrative selection of theinventive thought and not a limitation of the subject matter of theinvention.

EXAMPLE 1 HGEGTFTSDLSKQ-Nle-EEEAVRLFIEWLKNGR-NH₂ (SEQ ID NO.3) [Nle¹⁴,Arg³⁰]-exendin-4-(1-30)-NH₂

Example 1 was synthesized in a 0.02 mmol batch by the solid phase methodon 5-(4′-aminomethyl)-3′,5′-dimethoxyphenoxy)valerianyl-alanyl-aminomethyl-polystyrene(1%)divinylbenzene(loading: 0.5 mmol/g) on a multiple automated peptide synthesizer SyRoII from the MultiSyn Tech Company (Bochum). The α-amino functionalgroups of the amino acids were protected with 9-fluorenylmethoxycarbonyl(Fmoc). The side chain protective groups were t-butyl (tBu) for Asp,Glu, Ser and Thr, trityl (Trt) for Asn, Gln and His, t-butyloxycarbonyl(Boc) for Lys and Trp and 2,2,5,7,8-pentaethylchroman-6-sulfonyl (Pmc)for Arg. The protected amino acids were coupled sequentially in a10-fold excess using double couplings of 2 times 40 minutes duration andusing N,N-diisopropylcarbodiimide/1-hydroxybenzo-triazole as activationreagents. The peptide was cleaved from the polymeric support whilesimultaneously cleaving the protective groups in trifluoroacetic acid(85%) in the presence of 15% ethanedithiol/dimethylsulfide/m-cresol(1:1:1 v/v/v) for 120 minutes at room temperature. Subsequently thepeptide was precipitated with anhydrous diethyl ether and then washedseveral times with anhydrous diethyl ether to completely remove thethiols. Freeze-drying of the precipitate from water/tert. butanol (1:1)yielded 62 mg of the crude peptide. The crude peptide was purifiedwithin 30 minutes by reversed-phase HPLC with a gradient of 37% to 42%acetonitrile/0.9% TFA. The eluate was evaporated, lyophilized and gave ayield of 29 mg of a white solid with a purity of ≧97%.

Amino acid analysis; Ala 1,08 (1); Asx 1,91 (2); Glx 6,10 (6); Phe 1,78(2); Gly 3,10 (3); His 1,00 (1); Ile 0,88 (1); Lys 2,02 (2); Leu 3,24(3); Nle 1,10 (1); Arg 1,98 (2); Ser 2,04 (2); Thr 1,99 (2); Val 0,91(1); Trp 0,87 (1)

ESI-MS: 3488.2

EXAMPLE 2 HGEGTFTSDLSKQ-Nle-EEEAVRLFIEWLKNGY-NH₂ (SEQ ID No. 4) [Nle¹⁴,Tyr³⁰]-exendin-4-(1-30)-NH₂

Example 2 was synthesized in a 0.0076 mmol batch by the solid phasemethod on TentaGel® (Rapp Polymers, Tübingen) which was derivatized witha Rink-amide anchor (4-(2′,4′-dimethoxyphenyl-aminomethyl)-phenoxygroup) (loading: 0.18 mmol/g) on a multiple automated peptidesynthesizer SyRo II from the MultiSynTech Company (Bochum). Theprotected amino acids that were used were analogous to example 1, Theprotected amino acids were sequentially coupled in an eight-fold excesswith single couplings of 40 minutes duration at 40° C. and whilestirring. 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluroniumtetrafluoroborate (TBTU)/1-hydroxybenzotriazole were used as activationreagents with the addition of diisopropylethylamine. The peptide wascleaved and purified analogously to example 1. 18.1 mg of a while solidwith a purity of >95% was obtained.

Amino acid analysis: Ala 1,03 (1); Asx 1,90 (2); Glx 6,24 (6); Phe 1,94(2); Gly 3,12 (3); His 1,02 (1); Ile 1,09 (1); Lys 2,01 (2); Leu 3,06(3); Nle 1,08 (1); Arg 0,97 (1); Ser 1,98 (2); Thr 1,80 (2); Val 0,93(1); Trp 1,01 (1); Tyr 0,90 (1).

ESI-MS: 3494.8

EXAMPLE 3 HSDGTFTSDLSKQ-Nle-EEEAVRLFIEWLENGR-H₂ (SEQ ID No. 5) [Nle¹⁴,Arg³⁰]-exendin-3-(1-30)-NH₂

Example 3 was synthesized analogously to the method described forexample 2. 17.6 mg of a white solid with a purity of ≧99% was obtained.

Amino acid analysis: Ala 0,99 (1); Asx 2,98 (3); Glx 5,16 (5); Phe 2,08(2); Gly 2,16 (2); His 0,95 (1); Ile 1,03 (1); Lys 2,04 (2); Leu 2,91(3); Nle 1,05 (1); Arg 1,04 (1); Ser 3,00 (3); Thr 2,05 (2); Val 1,01(1); Trp 1,18 (1); Tyr 0,98 (1).

ESI-MS: 3504,4

EXAMPLE 4 HGEGTFTSDLSKQMEEEAVRLFIEWLKNGR-NH₂ (SEQ ID No. 6) [Arg³⁰]-exendin-4-(1-30)-NH₂

Example 4 was synthesized analogously to the method described forexample 1. 17.9 mg of a white solid with a purity of ≧96% was obtained.

Amino acid analysis: Ala 0,96 (1); Asx 2,01 (2); Glx 6,00 (6); Phe 1,80(2); Gly 3,21 (3); His 0,96 (1); Ile 1,07 (1); Lys 1,92 (2); Leu 2,98(3); Met 1,06 (1); Arg 1,90 (2); Ser 1,91 (2); Thr 2,09 (2); Val 0,97(1); Trp 0,84 (1).

ESI-MS: 3508,4

EXAMPLE 5 GEGTFTSDLSKQ-Nle-EEEAVRLFIEWLKNGR-NH₂ (SEQ ID No. 7) [Nle¹⁴,Arg³⁰]-exendin-4-(2-30)-NH₂

Example 5 was synthesized analogously to the method described forexample 2. 13.2 mg of a white solid with a purity of ≧97% was obtained.

Amino acid analysis: Ala 1,04 (1); Asx 1,98 (2); Glx 6,08 (6); Phe 1,86(2); Gly 2,91 (3); Ile 0,96 (1); Lys 1,84 (2); Leu 2,98 (3); Nle 1,04(1); Arg 1,90 (2); Ser 1,94 (2); Thr 1,92 (2); Val 0,96 (1); Trp 0,85(1).

ESI-MS: 3350,8

EXAMPLE 6 HGEGTFTSDLSKQMEEEAVRAFIEWLKNGR-NH₂ (SEQ ID No. 8) [Ala²¹,Arg³⁰]-exendin-4-(1-30)-NH₂

Example 6 was synthesized analogously to the method described forexample 1. 11.1 mg of a white solid with a purity of ≧95% was obtained.

Amino acid analysis: Ala 2,08 (2); Asx 1,93 (2); Glx 6,07 (6); Phe 1,74(2); Gly 2,97 (3); His 0,98 (1); Ile 0,87 (1); Lys 2,15 (2); Leu 2,02(2); Met 0,96 (1); Arg 2,13 (2); Ser 1,87 (2); Thr 2,07 (2); Val 1,04(1); Trp 0,87 (1).

ESI-MS: 3466,3

EXAMPLE 7 HGEGTFTSDLSKQMEEEAVRLFIEWLKAGR-NH₂ (SEQ ID No. 9) [Ala²⁸,Arg³⁰]-exendin-4-(1-30)-NH₂

Example 7 was synthesized analogously to the method described forexample 1. 15.0 mg of a white solid with a purity of ≧97% was obtained.

Amino acid analysis: Ala 1,98 (2); Asx 0,98 (1); Glx 6,22 (6); Phe 1,92(2); Gly 3,03 (3); His 0,99 (1); Ile 1,03 (1); Lys 2,05 (2); Leu 3,03(3); Met 0,96 (1); Arg 1,84 (2); Ser 1,98 (2); Thr 2,09 (2); Val 1,01(1); Trp 0,72 (1).

ESI-MS: 3465,4

EXAMPLE 8 HGEGTFTSDLSKQMEEEAVRAFIEWLKAGR-NH₂ (SEQ ID No. 10)[Ala^(21,28), Arg³⁰]-exendin-4-(1-30)-NH₂

Example 8 was synthesized analogously to the method described forexample 1. 18.4 mg of a white solid with a purity of ≧95% was obtained.

Amino acid analysis: Ala 3,12 (3); Asx 0,99 (1); Glx 6,04 (6); Phe 1,80(2); Gly 3,00 (3): His 0,96 (1); Ile 1,02 (1); Lys 1,84 (2); Leu 1,97(2); Met 0,98 (1); Arg 2,03 (2); Ser 1,91 (2); Thr 1,88 (2); Val 0,99(1); Trp 0,99 (1).

ESI-MS: 3423,3

EXAMPLE 9

It was possible to prepare the following exendin derivatives in highpurity in an analogous manner. (Ex-4=exendin-4, Ex-3=exendin-3)

Exendin-Derivative Seq. Sequence [A¹⁴, R³⁰]-Ex-4-(1-30)—OH 11HGEGTFTSDLSKQAEEEAVRLFIEWLKNGR—OH Ac-[Ile¹⁴, R³⁰]-Ex-4-(1-30)—NH₂ 12Ac-HGEGTFTSDLSKQIleEEEAVRLFIEWLKNGR—NH₂ [Nle¹⁴]-Ex-4-(1-27)—NH₂ 13HGEGTFTSDLSKQNleEEEAVRLFIEWLK—NH₂ [A^(14,29), R³⁰]-Ex-3-(1-30)—NH₂ 14HSDGTFTSDLSKQAEEEAVRLFIEWLKNAR—NH₂ [A^(14,27), R³⁰]-Ex-3-(1-30)—NH₂ 15HSDGTFTSDLSKQAEEEAVRLFIEWLANGR—NH₂ [A^(14,26), R³⁰]-Ex-3-(1-30)—NH₂ 16HSDGTFTSDLSKQAEEEAVRLFIEWAKNGR—NH₂ [A², Nle¹⁴, R³⁰]-Ex-4-(1-30)—NH₂ 17HAEGTFTSDLSKQNleEEEAVRLPIEWLKNGR—NH₂ [C², Nle¹⁴, R³⁰]-Ex-4-(1-30)—NH₂ 18HCEGTFTSDLSKQNleEEEAVRLFIEWLKNGR—NH₂ [D², Nle¹⁴, R³⁰]-Ex-4-(1-30)—NH₂ 19HDEGTFTSDLSKQNleEEEAVRLFIEWLKNGR—NH₂ [E², Nle¹⁴, R³⁰]-Ex-4-(1-30)—NH₂ 20HEEGTFTSDLSKQNleEEEAVRLFIEWLKNGR—NH₂ [F², Nle¹⁴, R³⁰]-Ex-4-(1-30)—NH₂ 21HPEGTFTSDLSKQNleEEEAVRLFIEWLKNGR—NH₂ [H², Nle¹⁴, R³⁰]-Ex-4-(1-30)—NH₂ 22HHEGTFTSDLSKQNleEEEAVRLFIEWLKNGR—NH₂ [I², Nle¹⁴, R³⁰]-Ex-4-(1-30)—NH₂ 23HIEGTFTSDLSKQNleEEEAVRLFIEWLKNGR—NH₂ [K², Nle¹⁴, R³⁰]-Ex-4-(1-30)—NH₂ 24HKEGTFTSDLSKQNleEEEAVRLFIEWLKNGR—NH₂ [L², Nle¹⁴, R³⁰]-Ex-4-(1-30)—NH₂ 25HLEGTFTSDLSKQNleEEEAVRLFIEWLKNGR—NH₂ [M², Nle¹⁴, R³⁰]-Ex-4-(1-30)—NH₂ 26HMEGTFTSDLSKQNleEEEAVRLFIEWLKNGR—NH₂ [N², Nle¹⁴, R³⁰]-Ex-4-(1-30)—NH₂ 27HNEGTFTSDLSKQNleEEEAVRLFIEWLKNGR—NH₂ [P², Nle¹⁴, R³⁰]-Ex-4-(1-30)—NH₂ 28HPEGTFTSDLSKQNleEEEAVRLFIEWLKNGR—NH₂ [Q², Nle¹⁴, R³⁰]-Ex-4-(1-30)—NH₂ 29HQEGTFTSDLSKQNleEEEAVRLFIEWLKNGR—NH₂ [R², Nle¹⁴, R³⁰]-Ex-4-(1-30)—NH₂ 30HREGTFTSDLSKQNleEEEAVRLFIEWLKNGR—NH₂ [S², Nle¹⁴, R³⁰]-Ex-4-(1-30)—NH₂ 31HSEGTFTSDLSKQNleEEEAVRLFIEWLKNGR—NH₂ [T², Nle¹⁴, R³⁰]-Ex-4-(1-30)—NH₂ 32HTEGTFTSDLSKQNleEEEAVRLFIEWLKNGR—NH₂ [V², Nle¹⁴, R³⁰]-Ex-4-(1-30)—NH₂ 33HVEGTFTSDLSKQNleEEEAVRLFIEWLKNGR—NH₂ [W², Nle¹⁴, R³⁰]-Ex-4-(1-30)—NH₂ 34HWEGTFTSDLSKQNleEEEAVRLFIEWLKNGR—NH₂ [Y², Nle¹⁴, R³⁰]-Ex-4-(1-30)—NH₂ 35HYEGTFTSDLSKQNleEEEAVRLFIEWLKNGR—NH₂ [A^(2,24), G¹⁶, E²¹, K^(20,28),Q¹⁷, R³⁰, S¹², V²⁷, Y¹³]-Ex-3-(1-30)—NH₂ 36HADGTFTSDLSSYMEGQAVKEFIAWLVKGR—NH₂ [A^(14,25), R³⁰]-Ex-3-(1-30)—NH₂ 37HSDGTFTSDLSKQAEEEAVRLFIEALKNGR—NH₂ [E³, A¹⁴, R³⁰]-Ex-3-(1-30)—NH₂ 38HSEGTFTSDLSKQAEEEAVRLFIEWLKNGR—NH₂ [A¹, V¹⁴, R³⁰]-Ex-3-(1-30)—NH₂ 39ASDGTFTSDLSKQVEEEAVRLFIEWLKNGR—NH₂ [A^(3,14), R³⁰]-Ex-4-(1-30)—NH₂ 40HGAGTFTSDLSKQAEEEAVRLFIEWLWNGR—NH₂ [A^(5,14), R³⁰]-Ex-4-(1-30)—NH₂ 41HGEGAFTSDLSKQAEEEAVRLFIEWLKNGR—NH₂ [A¹⁴, R³⁰, Y⁵]-Ex-4-(1-30)—NH₂ 42HGEGYFTSDLSKQAEEEAVRLFIEWLKNGR—NH₂ [A¹⁴, R³⁰, Y⁶]-Ex-4-(1-30)—NH₂ 43HGEGTYTSDLSKQAEEEAVRLFIEWLKNGR—NH₂ [A¹⁴, I⁶, R³⁰]-Ex-4-(1-30)—NH₂ 44HGEGTITSDLSKQAEEEAVRLFIEWLKNGR—NH₂ [A¹⁴, R³⁰, S⁷]-Ex-4-(1-30)—NH₂ 45HGEGTFSSDLSKQAEEEAVRLFIEWLKNGR—NH₂ [A¹⁴, R³⁰, Y⁷]-Ex-4-(1-30)—NH₂ 46HGEGTFYSDLSKQAEEEAVRLFIEWLKNGR—NH₂ [A¹⁴, R³⁰, T⁸]-Ex-4-(1-30)—NH₂ 47HGEGTFTTDLSKQAEEEAVRLPIEWLKNGR—NH₂ [A¹⁴, R³⁰, Y⁸]-Ex-4-(1-30)—NH₂ 48HGEGTFTYDLSKQAEEEAVRLFIEWLKNGR—NH₂ [A¹⁴, E⁹, R³⁰]-Ex-4-(1-30)—NH₂ 49HGEGTFTSELSKQAEEEAVRLFIEWLKNGR—NH₂ [A^(10,14), R³⁰]-Ex-4-(1-30)—NH₂ 50HGEGTFTSDASKQAEEEAVRLFIEWLKNGR—NH₂ [A^(11,14), R³⁰]-Ex-4-(1-30)—NH₂ 51HGEGTFTSDLAKQAEEEAVRLFIEWLKNGR—NH₂ [A^(12,14), R³⁰]-Ex-4-(1-30)—NH₂ 52HGEGTFTSDLSAQAEEEAVRLFIEWLKNGR—NH₂ [A^(13,14), R³⁰]-Ex-4-(1-30)—NH₂ 53HGEGTFTSDLSKAAEEEAVRLFIEWLKNGR—NH₂ [A^(14,15), R³⁰]-Ex-4-(1-30)—NH₂ 54HGEGTFTSDLSKQAAEEAVRLFIEWLKNGR—NH₂ [A^(14,16), G¹, R³⁰,S⁵]-Ex-3-(1-30)—NH₂ 55 GSDGSFTSDLSKQAEAEAVRLFIEWLWNGR—NH₂ [A^(14,17),K¹, R³⁰]-Ex-4-(1-30)—NH₂ 56 KGEGTFTSDLSKQAEEAAVRLFIEWLKNGR—NH₂ [A¹⁴,L¹⁸, R³⁰]-Ex-4-(1-30)—NH₂ 57 HGEGTFTSDLSKQAEEELVRLFIEWLKNGR—NH₂ [A¹⁴,I¹⁹, R³⁰]-Ex-4-(1-30)—NH₂ 58 HGEGTFTSDLSKQAEEEAIRLFIEWLKNGR—NH₂[A^(14,20), R³⁰]-Ex-4-(1-30)—NH₂ 59 HGEGTFTSDLSKQAEEEAVALFIEWLKNGR—NH₂[A¹⁴, R³⁰, Y²²]-Ex-3-(1-30)—NH₂ 60 HSDGTFTSDLSKQAEEEAVRLYIEWLKNGR—NH₂[A¹⁴, R³⁰, V²³]-Ex-4-(1-30)—NH₂ 61 HGEGTFTSDLSKQAEEEAVRLFVEWLKNGR—NH₂[A¹⁴, L²⁴, R³⁰]-Ex-4-(1-30)—NH₂ 62 HGEGTFTSDLSKQAEEEAVRLFILWLKNGR—NH₂[A^(14,25), R³⁰]-Ex-4-(1-30)—NH₂ 63 HGEGTFTSDLSKQAEEEAVRLFIEALKNGR—NH₂[A^(14,26), R³⁰]-Ex-4-(1-30)—NH₂ 64 HGEGTFTSDLSKQAEEEAVRLFIEWAKNGR—NH₂[A^(14,27), R³⁰]-Ex-4-(1-30)—NH₂ 65 HGEGTFTSDLSKQAEEEAVRLFIEWLANGR—NH₂[A^(14,29), R³⁰]-Ex-4-(1-30)—NH₂ 66 HGEGTFTSDLSKQAEEEAVRLFIEWLKNAR—NH₂[A¹⁴, R³⁰]-Ex-4-(1-30)—NH₂ 67 HGEGTFTSDLSKQAEEEAVRLFIEWLKNGR—NH₂[R³⁰]-Ex-3-(1-30)—NH₂ 68 HSDGTFTSDLSKQMEEEAVRLFIEWLKNGR—NH₂ [Nle¹⁴,Y³⁰]-Ex-3-(1-30)—NH₂ 69 HSDGTFTSDLSKQNleEEEAVRLFIEWLKNGY—NH₂ [Nle¹⁴,R³⁰]-Ex-3-(1-30)—OH 70 HSDGTFTSDLSKQNleEEEAVRLFIEWLKNGR—OH [A¹⁴,R³⁰]-Ex-3-(1-30)—NH₂ 71 HSDGTFTSDLSKQAEEEAVRLFIEWLKNGR—NH₂ [Nla¹⁴,R³⁰]-Ex-3-(2-30)—NH₂ 72 SDGTFTSDLSKQNleEEEAVRLFIEWLKNGR—NH₂ [Nle¹⁴,R³⁰]-Ex-3-(3-30)—NH₂ 73 DGTFTSDLSKQNleEEEAVRLFIEWLKNGR—NH₂ Ac-[Nle¹⁴,R³⁰]-Ex-3-(2-30)—NH₂ 74 Ac-DGTFTSDLSKQNleEEEAVRLFIEWLKNGR—NH₂ Ac-[Nle¹⁴,R³⁰]-Ex-3-(3-30)—NH₂ 75 Ac-DGTFTSDLSKQNleEEEAVRLFIEWLKNGR—NH₂[Nle¹⁴]-Ex-3-(1-27)—NH₂ 76 HSDGTFTSDLSKQNleEEEAVRLFIELK—NH₂ [K², P³,A¹⁴, R³⁰]-Ex-3-(1-30)—NH₂ 77 HKPGTFTSDLSKQAEEEAVRLFIEWLKNGR—NH₂ [A²,Nle¹⁴, R³⁰]-Ex-3-(1-30)—NH₂ 78 HADGTFTSDLSKQNleEEEAVRLFIEWLKNGR—NH₂ [C²,Nle¹⁴, R³⁰]-Ex-3-(1-30)—NH₂ 79 HCDGTFTSDLSKQNleEEEAVRLFIEWLKNGR—NH₂ [D²,Nle¹⁴, R³⁰]-Ex-3-(1-30)—NH₂ 80 HDDGTFTSDLSKQNleEEEAVRLFIEWLKNGR—NH₂ [E²,Nle¹⁴, R³⁰]-Ex-3-(1-30)—NH₂ 81 HEDGTFTSDLSKQNleEEEAVRLFIEWLKNGR—NH₂ [F²,Nle¹⁴, R³⁰]-Ex-3-(1-30)—NH₂ 82 HFDGTFTSDLSKQNleEEEAVRLFIEWLKNGR—NH₂ [G²,Nle¹⁴, R^(30]-Ex-3-(1-30)—NH) ₂ 83 HGDGTFTSDLSKQNleEEEAVRLFIEWLKNGR—NH₂[H² Nle¹⁴R³⁰]-Ex-3-(1-30)—NH₂ 84 HHDGTFTSDLSKQNleEEEAVRLFIEWLKNGR—NH₂[I², Nle¹⁴, R³⁰]-Ex-3-(1-30)—NH₂ 85 HIDGTFTSDLSKQNleEEEAVRLFIEWLKNGR—NH₂[K², Nle¹⁴, R³⁰]-Ex-3-(1-30)—NH₂ 86 HKDGTFTSDLSKQNleEEEAVRLFIEWLKNGR—NH₂[L², Nle¹⁴, R³⁰]-Ex-3-(1-30)—NH₂ 87 HLDGTFTSDLSKQNleEEEAVRLFIEWLKNGR—NH₂[M², Nle¹⁴, R³⁰]-Ex-3-(1-30)—NH₂ 88 HMDGTFTSDLSKQNleEEEAVRLFIEWLKNGR—NH₂[N²Nle¹⁴, R³⁰]-Ex-3-(1-30)—NH₂ 89 HNDGTFTSDLSKQNleEEEAVRLFIEWLKNGR—NH₂[P², Nle¹⁴, R³⁰]-Ex-3-(1-30)—NH₂ 90 HPDGTFTSDLSKQNleEEEAVRLFIEWLKNGR—NH₂[Q², Nle¹⁴, R³⁰]-Ex-3-(1-30)—NH₂ 91 HQDGTFTSDLSKQNleEEEAVRLFIEWLKNGR—NH₂[R², Nle¹⁴, R³⁰]-Ex-3-(1-30)—NH₂ 92 HRDGTFTSDLSKQNleEEEAVRLFIEWLKNGR—NH₂[T², Nle¹⁴, R³⁰]-Ex-3-(1-30)—NH₂ 93 HTDGTFTSDLSKQNleEEEAVRLFIEWLKNGR—NH₂[V², Nle¹⁴, R³⁰]-Ex-3-(1-30)—NH₂ 94 HVDGTFTSDLSKQNleEEEAVRLFIEWLKNGR—NH₂[W², Nle¹⁴, R³⁰]-Ex-3-(1-30)—NH₂ 95 HWDGTFTSDLSKQNleEEEAVRLFIEWLKNGR—NH₂[Y², Nle¹⁴, R³⁰]-Ex-3-(1-30)—NH₂ 96 HYDGTFTSDLSKQNleEEEAVRLFIKWLKNGR—NH₂[A^(3,14), R³⁰]-Ex-3-(1-30)—NH₂ 97 HSAGTFTSDLSKQAEEEAVRLFIEWLKNGR—NH₂[A^(5,14), R³⁰]-Ex-3-(1-30)—NH₂ 98 HSDGAFTSDLSKQAEEEAVRLFIEWLKNGR—NH₂[A¹⁴, R³⁰, Y⁵]-Ex-3-(1-30)—NH₂ 99 HSDGYFTSDLSKQAEEEAVRLFIEWLKNGR—NH₂[A¹⁴, R³⁰, Y⁶]-Ex-3-(1-30)—NH₂ 100 HSDGTYTSDLSKQAEEEAVRLFIEWLKNGR—NH₂[A¹⁴, I⁶, R³⁰]-Ex-3-(1-30)—NH₂ 101 HSDGTITSDLSKQAEEEAVRLFIEWLKNGR—NH₂[A¹⁴, R³⁰, S⁷]-Ex-3-(1-30)—NH₂ 102 HSDGTFSSDLSKQAEEEAVRLFIEWLKNGR—NH₂[A¹⁴, R³⁰, Y⁷]-Ex-3-(1-30)—NH₂ 103 HSDGTFYSDLSKQAEEEAVRLFIEWLKNGR—NH₂[A¹⁴, R³⁰, T⁸]-Ex-3-(1-30)—NH₂ 104 HSDGTFTTDLSKQAEEEAVRLFIEWLKNGR—NH₂[A¹⁴, R³⁰, Y⁸]-Ex-3-(1-30)—NH₂ 105 HSDGTFTYDLSKQAEEEAVRLFIEWLKNGR—NH₂[A¹⁴, E⁹, R³⁰]-Ex-3-(1-30)—NH₂ 106 HSDGTFTSELSKQAEEEAVRLFIEWLKNGR—NH₂[A^(10,14), R³⁰]-Ex-3-(1-30)—NH₂ 107 HSDGTFTSDASKQAEEEAVRLFIEWLKNGR—NH₂[A^(11,14), R³⁰]-Ex-3-(1-30)—NH₂ 108 HSDGTFTSDLAKQAEEEAVRLFIEWLKNGR—NH₂[A^(12,14), R³⁰]-Ex-3-(1-30)—NH₂ 109 HGEGTFTSDLSAQAEEEAVRLFIEWLKNGR—NH₂[A^(13,14), R³⁰]-Ex-3-(1-30)—NH₂ 110 HSDGTFTSDLSKAAEEEAVRLFIEWLKNGR—NH₂[A^(14,15), R³⁰]-Ex-3-(1-30)—NH₂ 111 HSDGTFTSDLSKQAAEEAVRLFIEWLKNGR—NH₂[A^(14,17), K¹, R³⁰]-Ex-3-(1-30)—NH₂ 112KSDGTFTSDLSKQAEEAAVRLFIEWLKNGR—NH₂ [A¹⁴, L¹⁸, R³⁰]-Ex-3-(1-30)—NH₂ 113HSDGTFTSDLSKQAEEELVRLFIEWLKNGR—NH₂ [A¹⁴, I¹⁹, R³⁰]-Ex-3-(1-30)—NH₂ 114HSDGTFTSDLSKQAEEEAIRLFIEWLKNGR—NH₂ [A^(14,20), R³⁰]-Ex-3-(1-30)—NH₂ 115HSDGTFTSDLSKQAEEEAVALFIEWLKNGR—NH₂ [A¹⁴, R³⁰, Y²²]-Ex-3-(1-30)—NH₂ 116HSDGTFTSDLSKQAEEEAVRLYIEWLKNGR—NH₂ [A¹⁴, R³⁰, V²³]-Ex-3-(1-30)—NH₂ 117HSDGTFTSDLSKQAEEEAVRLFVEWLKNGR—NH₂ [A¹⁴, L²⁴, R³⁰]-Ex-3-(1-30)—NH₂ 118HSDGTFTSDLSKQAEEEAVRLFILWLKNGR—NH₂ Suc-[Nle¹⁴, R³⁰]-Ex-3-(3-30)—NH₂ 119Suc-DGTFTSDLSKQNleEEEAVRLFIEWLKNGR—NH₂ [Nle¹⁴, R³⁰]-Ex-3-(1-30)—NH₂ 120HSDGTFTSDLSKQNleEEEAVRLFIEWLKNGR—NH₂

The following examples were synthesized in a 0.02 mmol batch by thesolid phase method on RAM resin® (Rapp Polymere, Tübingen) in whichaminomethylpolystyrene(1%)divinylbenzene is derivatized with Rink amideanchor (4-(2′,4′-dimethoxyphenyl-aminomethyl)-phenoxy group) (loading:0.5 mmol/g). The syntheses were carried out on a multiple automatedpeptide synthesizer SyRo II from the MultiSynTech Company (Bochum). Theprotected amino acids that were used were analogous to those ofexample 1. The protected amino acids were sequentially coupled in a5-fold excess with single couplings of 40 minutes duration at 40° C. andwhile stirring. 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluroniumtetrafluoroborate (TBTU) with the addition of diisopropylethylamine wasused as the activation reagent. The cleavage and purification of thepeptide was carried out analogously to example 1. The yields, puritiesand analytical data of the peptides synthesized as described above arelisted in the following two tables.

TABLE 1 Yield Purity Seq. ID [mg] [%] ESI-MS 36 6.0 99 3343.3 38 12.8 993477.4 68 14.2 99 3523.4 69 14.1 >99 3512.1 70 18.0 95 3506.0 71 22.4 953463.6 72 6.6 >99 3368.0 73 17.8 >99 3281.1 75 12.0 99 3323.1 76 14.0 993178.1 77 7.2 99 3486.6 78 23.8 95 3488.1 79 19.0 95 3520.1 80 15.2 953531.9 81 8.6 >99 3545.9 82 23.6 98 3563.9 83 25.4 95 3475.6 84 10.4 >993554.1 85 8.2 >99 3530.1 86 10.4 95 3545.3 87 9.2 >99 3530.1 88 13.6 >993548.1 89 10.6 >99 3531.0 90 9.4 96 3514.9 91 6.0 >99 3545.4 92 15.4 >993574.9 93 10.1 >99 3519.7 94 9.4 >99 3517.7 95 12.0 >99 3604.7 96 9.8 953581.8 120 8.5 95 3505.6

TABLE 2 Amino acid analyses Seq. ID Ala Arg Asx Glx Gly His Ile Leu LysNle Phe Ser Thr Trp Val  36^(a) 3.18 0.99 1.95 3.04 2.94 0.89 1.03 2.142.09 1.95 3.06 1.88 0.99 1.94 (3) (1) (2) (3) (3) (1) (1) (2) (2) (2)(3) (2) (1) (2)  38 2.08 2.10 2.00 6.06 2.04 0.81 1.01 3.25 2.13 1.963.08 1.82 0.94 1.02 (2) (2) (2) (6) (2) (1) (1) (3) (2) (2) (3) (2) (1)(1)  68^(b) 1.02 2.14 3.16 5.06 1.71 0.93 1.02 3.21 2.08 1.93 3.00 1.880.98 1.02 (1) (2) (3) (5) (2) (1) (1) (3) (2) (2) (3) (2) (1) (1) 69^(c) 1.02 1.12 2.99 4.94 1.81 0.77 1.12 3.26 2.12 1.04 1.99 2.91 1.811.06 1.03 (1) (1) (3) (5) (2) (1) (1) (3) (2) (1) (2) (3) (2) (1) (1) 70 1.08 2.15 2.96 5.07 1.84 0.85 1.06 3.23 2.06 0.92 1.99 2.01 1.841.05 1.04 (1) (2) (3) (5) (2) (1) (1) (3) (2) (1) (2) (3) (2) (1) (1) 71 1.99 2.06 3.14 4.98 1.95 0.85 1.01 3.20 2.08 1.94 3.09 1.90 1.030.96 (2) (2) (3) (5) (2) (1) (1) (3) (2) (2) (3) (2) (1) (1)  72 1.022.12 3.06 5.02 1.94 1.02 3.25 2.11 0.95 1.98 2.92 1.88 1.02 1.03 (1) (2)(3) (5) (2) (1) (3) (2) (1) (2) (3) (2) (1) (1)  73 1.01 2.04 3.02 5.031.83 0.99 3.19 2.07 1.06 1.98 2.00 1.87 0.97 1.03 (1) (2) (3) (5) (2)(1) (3) (2) (1) (2) (2) (2) (1) (1)  75 1.09 2.21 3.10 5.18 1.75 1.163.26 2.10 1.08 1.95 2.10 2.04 1.28 0.88 (1) (2) (3) (5) (2) (1) (3) (2)(1) (2) (2) (2) (1) (1)  76 1.03 1.06 2.05 4.90 0.94 0.88 1.03 3.18 2.011.03 1.95 3.02 1.86 0.98 1.03 (1) (1) (2) (5) (1) (1) (1) (3) (2) (1)(2) (3) (2) (1) (1)  77^(d) 2.07 2.00 2.10 5.07 1.78 0.93 1.01 3.14 3.121.93 2.05 1.93 1.01 1.01 (2) (2) (2) (5) (2) (1) (1) (3) (3) (2) (2) (2)(1) (1)  78 2.11 2.17 3.14 5.01 1.92 0.90 1.06 3.25 2.06 1.05 1.99 2.061.90 1.00 1.05 (2) (2) (3) (5) (2) (1) (1) (3) (2) (1) (2) (2) (2) (1)(1)  79^(e) 1.16 2.10 3.07 5.07 1.78 0.66 1.08 3.30 2.06 0.96 1.93 2.021.87 1.06 1.05 (1) (2) (3) (5) (2) (1) (1) (3) (2) (1) (2) (2) (2) (1)(1)  80 1.07 2.13 3.78 5.03 1.82 0.84 1.05 3.20 2.04 0.92 1.97 1.99 1.821.05 1.03 (1) (2) (4) (5) (2) (1) (1) (3) (2) (1) (2) (2) (2) (1) (1) 81 1.04 2.06 3.08 5.96 2.01 0.82 1.02 3.17 2.08 0.95 2.04 2.05 1.891.01 0.96 (1) (2) (3) (6) (2) (1) (1) (3) (2) (1) (2) (2) (2) (1) (1) 82 1.09 1.96 3.01 4.99 1.89 1.02 1.02 3.14 2.04 0.91 2.74 1.98 1.850.98 0.99 (1) (2) (3) (5) (2) (1) (1) (3) (2) (1) (3) (2) (2) (1) (1) 83 1.08 2.22 3.09 5.01 2.85 0.83 1.11 3.23 2.13 1.12 1.99 2.09 1.860.99 1.03 (1) (2) (3) (5) (3) (1) (1) (3) (2) (1) (2) (2) (2) (1) (1) 84 1.01 1.98 3.09 5.01 1.71 1.69 1.07 3.18 2.07 0.94 1.99 1.90 1.840.98 1.01 (1) (2) (3) (5) (2) (2) (1) (3) (2) (1) (2) (2) (2) (1) (1) 85 1.07 2.12 3.11 5.03 1.88 0.93 1.83 3.23 2.11 1.11 1.97 2.03 1.940.97 1.02 (1) (2) (3) (5) (2) (1) (2) (3) (2) (1) (2) (2) (2) (1) (1) 86 1.03 2.11 3.07 5.03 2.10 0.88 1.11 3.21 3.00 0.98 1.97 2.05 1.881.01 0.99 (1) (2) (3) (5) (2) (1) (1) (3) (3) (1) (2) (2) (2) (1) (1) 87 1.11 2.16 3.13 5.03 1.94 0.89 1.09 4.05 2.23 0.93 1.97 2.07 1.920.95 1.00 (1) (2) (3) (5) (2) (1) (1) (4) (2) (1) (2) (2) (2) (1) (1) 88^(f) 1.03 2.14 3.16 5.07 1.71 0.93 1.02 3.21 2.09 0.94 1.93 2.01 1.880.99 1.02 (1) (2) (3) (5) (2) (1) (1) (3) (2) (1) (2) (2) (2) (1) (1) 89 1.08 1.97 3.89 5.06 1.95 0.88 1.03 3.19 2.08 0.92 1.94 2.03 1.870.99 0.97 (1) (2) (4) (5) (2) (1) (1) (3) (2) (1) (2) (2) (2) (1) (1) 90^(g) 1.00 2.09 3.18 5.04 1.97 0.86 1.02 3.23 2.10 1.01 1.96 2.09 1.921.04 0.97 (1) (2) (3) (5) (2) (1) (1) (3) (2) (1) (2) (2) (2) (1) (1) 91 1.04 2.08 3.07 5.73 1.87 0.80 1.05 3.21 2.10 1.05 1.93 1.95 1.791.11 1.06 (1) (2) (3) (6) (2) (1) (1) (3) (2) (1) (2) (2) (2) (1) (1) 92 1.01 3.11 3.02 5.03 1.83 0.82 0.99 3.19 2.07 1.06 1.97 2.00 1.870.97 1.03 (1) (3) (3) (5) (2) (1) (1) (3) (2) (1) (2) (2) (2) (1) (1) 93 1.05 2.12 3.02 5.04 1.98 0.82 1.02 3.28 2.14 1.00 1.96 2.06 2.750.95 1.03 (1) (2) (3) (5) (2) (1) (1) (3) (2) (1) (2) (2) (3) (1) (1) 94 1.07 2.17 3.04 5.08 1.72 0.82 1.14 3.20 2.06 1.06 1.92 2.06 2.001.26 1.72 (1) (2) (3) (5) (2) (1) (1) (3) (2) (1) (2) (2) (2) (1) (2) 95 1.05 2.18 3.04 5.04 1.84 0.80 1.04 3.31 2.14 0.95 1.96 2.02 1.881.76 1.14 (1) (2) (3) (5) (2) (1) (1) (3) (2) (1) (2) (2) (2) (2) (1) 96^(h) 1.03 2.25 3.03 5.00 1.84 0.78 1.14 3.30 2.15 1.05 2.02 1.96 1.841.07 1.05 (1) (2) (3) (5) (2) (1) (1) (3) (2) (1) (2) (2) (2) (1) (1)120 1.11 1.98 3.05 5.06 1.91 1.03 1.03 3.18 2.07 0.93 1.95 2.94 1.880.99 1.00 (1) (2) (3) (5) (2) (1) (1) (3) (2) (1) (2) (3) (2) (1) (1)^(a)methionine 0.96 (1); tyrosine 0.86 (1) ^(b)methionine 0.94 (1)^(c)tyrosine 0.82 (1) ^(d)proline 1.02 (1) ^(e)cysteine could not bedetected under the given hydrolysis conditions ^(f)methionine 0.93 (1)^(g)proline 0.86 (1) ^(h)tyrosine 0.87 (1)

EXAMPLE 10 Pharmacological Data

Peptide Metabolism in Ectopeptidase Preparations or in Kidney MicrovilliMembrane Preparations

Background

A group of ectopeptidases is responsible for the post-secretorymetabolism of peptide hormones. These enzymes are bound to the plasmamembranes of various cell types. Their active site is oriented towardsthe extracellular space. In addition these enzymes are present in highconcentrations in the brush border membranes of the proximal renaltubuli. Renal brush border microvilli membranes (BBM) are therefore asuitable source for the relevant ectopeptidases and can be used as an invitro test for the metabolic stability of synthetic peptides.Alternatively it is possible to use ectopeptidase preparations. Thehuman neutral endopeptidase 24.11 as well as dipeptidyl peptidase IVwere used as examples since GLP-1 is a substrate of both theseectopeptidases.

Preparation of Brush Border Microvilli Membranes

Microvilli membranes of the rat and pig kidney cortex are isolated bymeans of subcellular fractionation using the differential centrifugationmethod (Booth and Kenny (1975)). 4 Brush border ectopeptidases areexamined fluorimetrically and other marker enzymes are measuredcolorimetrically in order to assess the degree of purity and the yieldof membranes.

Ectopeptidase Preparations

Purified human neutral endopeptidase 24.11 was obtained in a recombinantform from Genentech (San Francisco, USA), dipeptidyl peptidase IV wasobtained as an isolate from human placenta from Calbiochem (Bad Soden).

Incubation Protocol

Microvilli membranes (0.5-1 μg protein) or the respective ectopeptidasepreparation (60-300 ng) were incubated with 10 μg peptide (about 3 nmol)in 100 μl HEPES buffer (50 mM, pH 7.4) which contained 50 mM NaCl. Thereactions were terminated at predetermined times (duration up to 1 hour)by boiling. Subsequently the samples were centrifuged (10,000×g),diluted with 150 μl 0.1% TFA and analysed by means of reversed phase(RP) HPLC. Each sample was determined in duplicate.

HPLC Analysis

A system with the following components was used for HPLC analysis: A2248 low pressure pump (Pharmacia-LKB, Freiburg), a WISP 10Bautoinjector (Millipore-Waters, Eschborn), a UV detector SP-4 (Gynkotec,Berlin), a low pressure mixing system (Pharmacia-LKB, Freiburg) and aprogram manager software control (Pharmacia-LKB, Freiburg). Theseparations were carried out over Lichrospher C-8, 5 μ, 4×124 mm (Merck,Darmstadt) with a binary gradient using the mobile solvents A: 0.1%trifluoroacetic acid (TFA) and B: acetonitrile:water:TFA (70:29:0.1).After injection of 244 μl of the sample solution onto a columnequilibrated with the motile solvent A, the incubation products wereeluted with a linear gradient of 0% to 80% B in 80 min and detected at215 nm UV absorption.

Calculation of the Proteolysis Rates

Two measurements were carried out for each incubation period of eachpeptide and the average peak height of the substrate peak was plottedversus time. Using GLP-1 as an example it was possible to show that thepeak height is linearly proportional to the quantity of the peptide inthe sample solution. Furthermore a linear decrease of the peak heightwith time was observed within the first hour of incubation with themicrovilli membranes or the peptidases. Hence the proteolysis rate isdetermined from the decrease in the height of the substrate peak and isexpressed as [μmol substrate/mg protein/minute].

Degradation Stability of Exendin Analogues

Incubation With Human Neutral Endopeptidase 24.11b

[Nle¹⁴,Arg³⁰]-exendin-4-(1-30)-NH₂ (SEQ ID No. 3) was incubated with theneutral endopeptidase 24.11 as described above and the degradation ratewas determined. GLP-1-(7-36)-NH₂ served as a control. The results areshown in Table 3.

TABLE 3 Incubations with dipeptidyl peptidase IV Degradation rate[mM/100 ng/ml NEP24.11/min] GLP1-(7-36)-NH₂ 0.0586[Nle¹⁴,Arg³⁰]-Ex-4-(1-30)-NH₂ 0.0083 Exmp. 1

The peptides listed in Table 4 were incubated with dipeptidyl peptidaseIV (DDP-IV) as described above. Each of the incubations was terminatedat the time when the GLP-1-(7-36)-NH₂ exhibited 50% hydrolysis. Thesubstrate peak of each peptide was collected from the rpHPLC run andexamined by mass spectroscopy in order to exclude truncated products.

TABLE 4 Incubations with brush border microvilli membranes SubstrateAnalogue Seq. ID for DDP-IV [Ala²,Nle¹⁴,Arg³⁰]-Ex-3-(1-30)-NH₂ 78 noproteolysis GLP1-(7-36)-NH₂ 50% proteolysis

The proteolysis rates which were calculated after incubation with brushborder microvilli membranes (BBM) by the protocol described above areshown in Table 5. GLP-1-(7-36)-NH₂ served as a control.

TABLE 5 Insulin secretion by isolated islet cells Seq. Degradation rateAnalogue ID [ng peptide/min/mg BBM] GLP1-(7-36)-NH₂ 880.00[Lys²,Nle¹⁴,Arg³⁰]-Ex-3-(1-30)-NH₂ 86 2.05

Organ Removal

The abdomen of anaesthetised (0.3-0.5 ml nembutal/isotonic salinesolution 1:4 i.p.) mice is opened by a median incision and two sideincisions, the peritoneum is immobilized and cut open at the costal archalong the diaphragm. All organs are inflated and stained red byinjection of a neutral red solution into the left ventricle. Thepancreas is carefully removed along the stomach and duodenum up to themesenteries. Until digestion the pancreas is placed in an ice-cooledpetri dish in Hank's balanced salt solution (HBBS) and a few drops ofneutral red.

Islet Preparation

Two pancreases are dabbed with cellulose, placed in a tube, 5 ml freshlyprepared collagenase solution (collagenase (Cl. histolyticum) 0.74 U/mg,Serva, 2 mg/ml in HBBS/water 1:9, pH 7.4) is added and they areincubated for 18 minutes at 37° C. while shaking. Subsequently acentrifugation is carried out at 1000 rpm for 1 minute. The supernatantis discarded, In a second digestion step 5 ml collagenase solution (1mg/ml) is incubated for 4 minutes, shaken and undigested tissue issedimented. The supernatant is decanted and the whole process isrepeated four to five times. The supernatant is then centrifuged for 1minute at 1000 rpm and the collagenase solution is discarded. Theremaining pellet is shaken with ice-cold HBBS and sedimented for ca. 10minutes on ice. This wash process is repeated for a further three times.The faint pink stained islets are picked out from the washed pelletsunder a stereoscopic magnifying glass and transferred to culture medium(100 ml RPMI 1640 (Gibco), 1 ml glutamine, 1 ml penicillin, 1 mlCibrobay antibiotic (Bayer), 10 ml foetal calf serum, 2 ml Hepes buffer1 M). In order to obtain the purest possible culture, the islets arepicked two to three times and transferred to fresh culture medium.

Stimulation of the Islets

The islet cells from the culture medium are distributed in Eppendorffvessels containing 200 ml stimulation buffer (118 mM NaCl, 0.2 mMNaH²PO⁴, 0.565 mM MgCl², 1.25 mM CaCl², 4.1 mM KCl, 10 mM Hepes, 1% BSA,3.3 mM glucose; pH 7.4) in a quantity of 10 islets per Eppendorff vesseland placed in an incubator for 1 hour at 37° C. Subsequently thepeptides to be tested are added and filled up to 500 ml with stimulationbuffer and incubated for one hour at 37° C. The islets are centrifugedat 1000 rpm for 1 minute. The amount of C-peptide is measured in thesupernatant using an insulin-RIA (DPC Biermann, Nauheim). Each testsubstance was determined in quadruplicate.

Activity of the Exendin Analogues

Some exendin analogues were tested as described above on isolated isletcells for insulin secretory activity. The data are shown as an examplein the following table.

Insulin Release From Isolated Islets After 1 Hour [mIU/h/10 Islets] inthe Presence of 10 mM Glucose:

TABLE 6 Seq. Control GLP1-(7-36)-NH₂ ID 84 10 mM glucose 30.21 10⁻⁷ (10mM glucose) 53.52 48.94 10⁻⁸ (10 mM glucose) 42.78 41.72 10⁻⁹ (10 mMglucose) 29.99 38.76 10⁻¹⁰ (10 mM glucose) 35.05

Measurement of the Increase of the Cytosolic Calcium Concentration in BCells of the Endocrine Pancreas (Clonal B Cell Line INS-1)

Culture of INS-1-cells (Asfari, M., 1992):

INS-1 cells are cultured in RPMI 1640 medium containing 10% FCS, 10 mMHEPES buffer (pH 7.4), 2 mM L-glutamine, 100 i.U. penicillin/ml, 100 μgstreptomycine/ml, 1 mM pyruvate (sodium salt) and 50 μM2-mercaptoethanol at 37° C. in an atmosphere of 95% air and 5% CO₂.After 6 to 8 days growth on plastic cell culture plates, thesubconfluent cells, after rinsing once with PBS (phosphate-bufferedsaline), are detached from the base by incubating for four minutes at37° C. with 0.025% trypsin and 0.27 mM EDTA in isoosmotic salinesolution

Preparation of the Cells for Calcium Measurements:

The detached cells are resuspended in Spinner medium (culture medium asabove, but containing 5% FCS and 25 mM HEPES) and incubated for two anda half hours in a Spinner bottle with a stirrer bar. Afterwards themedium is removed by centrifugation and the cells are resuspended inSpinner medium. They are then incubated for 30 minutes at 37° C. with 2μM Fura-2/acetoxymethyl ester under the same conditions as before. TheFura loading of the cells is terminated by washing the cells onceSpinner medium (room temperature). Afterwards the cells are resuspendedin spinner medium at room temperature (2×10⁷ cells/ml). The cells arethen removed from this suspension for calcium measurements.

Measurements of the Cytosolic Calcium Concentration:

The measurements are carried out at 37° C. in a modified Krebs-Ringerbuffer (KRBH) containing 136 mM NaCl, 4.8 mM KCl, 2 mM CaCl₂, 1.2 MMMgSO₄, 1.2 mM KH₂PO₄, 5 mM NaNCO₃, 10 mM glucose, 250 μM sulfinpyrazone(to inhibit the Fura-2 efflux into the medium) and 25 mM HEPES buffer(adjusted to pH 7.4 with NaOH). The cell concentration is 1-2×10⁶/ml.The measurements are carried out in a cuvette stirred with a stirringbar in a spectrofluorimeter at 37° C. with 1.5 ml cell suspension. Theexcitation wavelength is 340 nm, the emission wavelength is 505 nm. Atthe end of the measurement 50 μM MnCl₂ is added which is followed by 100μm DTPA (diethylenetriamine pentaacetate) in order to determine theproportion of extracellular fluorescence indicator relative to themeasured fluorescence by temporarily quenching the fluorescence ofextracellular Fura. After the addition of DTPA the entire Fura isfirstly converted into a calcium-saturated and then into a calcium-freestate to determine the calibration values F_(max) (calcium-saturated)and F_(min) (calcium-free) for the respective measurement. For thispurpose the cells are lysed by adding 0.1% Triton X-100. The dye issaturated with calcium by contact with the high extracellular calciumconcentration. Subsequently 5 mM EGTA(ethylenebis(oxyethylenenitrilo)-tetraacetate) and 20 mM Tris solutionare added in order to completely convert the dye into the calcium-freeform.

The cytosolic calcium ion concentration is calculated according to thealgorithm introduced by R. Tsien and colleagues (Grynkiewicz, G., 1985):

[Ca²⁺]_(cyt)=((F−F _(min))/(F _(max) −F))×K _(D)

(F: fluorescence of the respective measurement point; KD: dissociationconstant of the calcium complex of Fura-2, 225 nM (Grynkiewicz, G.,1985)).

(Before this calculation a compensation is carried out for the presenceof extracellular Fura. For this the fluorescence quantity (extracellularFura) determined by manganese addition is firstly subtracted from thefluorescence values of the measurement points. Then F_(max) is correctedby substracting this quantity. Finally the correction value for F_(min)is determined. For this purpose the fluorescence quantity determined byaddition of manganese is divided by the value 2.24. The value 2.24 wasdetermined as the intrinsic instrument proportionality factor betweenthe fluorescence of calcium-saturated and calcium-free Fura-2 at anexcitation wavelength of 340 nm (measured with unesterified, freeFura-2). The correction value obtained in this manner was subtractedfrom F_(min)).

The examined peptides were added as 1000-fold concentrated solutions(10⁻⁵ M) in KRBH without CaCl₂ and glucose.

Activity of the Exendin Analogues

Several exendin analogues were tested in the calcium assay on INS-1cells as described above for their biolotical activity. The data areshown as an example in FIG. 1 as well as in Table 7.

TABLE 7 SEQ. Δ[Ca²⁺]cyt ± SD ID Nr. Concentration of the peptides 10⁻⁸ M(n = 4) 6 [Arg³⁰]-exendin-(1-30)-NH₂ 64 ± 8 nM  3[Nle¹⁴,Arg³⁰]-exendin-(1-30)-NH₂ 63 ± 8 nM  8[Ala²¹,Arg³⁰]-exendin-(1-30)-NH₂ 61 ± 11 nM 9[Ala²⁸,Arg³⁰]-exendin-(1-30)-NH₂ 65 ± 15 nM 10[Ala^(21,28),Arg³⁰]-exendin-(1-30)-NH₂ 69 ± 30 nM Control: GLP-1-(7-36)amide 65 ± 10 nM

Competition With GLP-1-(7-36)-NR₂ on B Cells of the Endocrine Pancreas(Clonal B Cell Line INS-1)

Culture of INS-1-cells (Asfari, M., 1992)

See measurement of calcium concentration

Competition Experiments

The detached cells are taken up and suspended in Krebs-Ringer buffer (25mM Tris, 120 mM NaCl, 1.2 mM MgSO₄, 5 mM KCl, 1 mM Na-EDTA, 15 mMCH₃COONa adjusted to pH 7.4 and supplemented with 1% HSA and 0.1%bacitracin). 250 ml is removed each time from this suspension for areaction mixture, admixed with 20 ml tracer (^(125I)-GLP1-(7-36)-NH₂,20,000 cpm) and 30 ml of the peptide to be examined in the correspondingdilution. Subsequently it is incubated for 30 minutes at 37° C.,centrifuged for 4 minutes at 13,000 rpm, washed three times with bufferand the radioactivity bound to the pellet (γ-counter) is measured.Competition curves were obtained by incubation with 10 differentdilutions of the peptide to be tested (10⁻¹⁰-10⁻⁶ M in Krebs-Ringerbuffer).

Receptor Affinity of the Exendin Analogues

The data are shown as an example in Table 8. GLP-1-(7-NH₂ served as astandard.

TABLE 8 Seq. Kd_(GLP1) ± SD Kd ± SD Kd/ ID peptide [nM] [nM] Kd_(GLP1)69 [Nle¹⁴,Tyr³⁰]-Ex3- 1.04 ± 0.05 0.56 ± 0.08 0.5 (1-30)-NH₂

Literatur

Albericio, F. and Barany, G. (1987) Int. J. Peptide Protein Res. 30,206-216.

Asfari, M., Janjic, D., Meda, P., Li, G., Halban, P. A. and Wollheim, C.B. (1992) Endocrinology 130, 167-178.

Barlos, K., Gatos, D., Kapolos, S., Paphotiu, G., Schafer, W., andWengqing, Y. (1989) Tetrahedron Lett. 30, 3947-3950.

Booth, and Kenny, (1975) Biochem. J. 142, 575-581.

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120 30 amino acids amino acid single strand linear peptide peptide 30/product= “Xaa denotes all amino acids except for Gly” 1 His Ser Asp GlyThr Phe Thr Ser Asp Leu Ser Lys Gln Met Glu Glu 1 5 10 15 Glu Ala ValArg Leu Phe Ile Glu Trp Leu Lys Asn Gly Xaa 20 25 30 30 amino acidsamino acid single strand linear peptide peptide 30 /product= “Xaadenotes all amino acids except for Gly” 2 His Gly Glu Gly Thr Phe ThrSer Asp Leu Ser Lys Gln Met Glu Glu 1 5 10 15 Glu Ala Val Arg Leu PheIle Glu Trp Leu Lys Asn Gly Xaa 20 25 30 30 amino acids amino acidsingle strand linear peptide peptide 14 /product= “Xaa denotes Nle” 3His Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Xaa Glu Glu 1 5 1015 Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Lys Asn Gly Arg 20 25 30 30amino acids amino acid single strand linear peptide peptide 14 /product=“Xaa denotes Nle” 4 His Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys GlnXaa Glu Glu 1 5 10 15 Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Lys AsnGly Tyr 20 25 30 30 amino acids amino acid single strand linear peptidepeptide 14 /product= “Xaa denotes Nle” 5 His Ser Asp Gly Thr Phe Thr SerAsp Leu Ser Lys Gln Xaa Glu Glu 1 5 10 15 Glu Ala Val Arg Leu Phe IleGlu Trp Leu Lys Asn Gly Arg 20 25 30 30 amino acids amino acid singlestrand linear peptide 6 His Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser LysGln Met Glu Glu 1 5 10 15 Glu Ala Val Arg Leu Phe Ile Glu Trp Leu LysAsn Gly Arg 20 25 30 29 amino acids amino acid single strand linearpeptide peptide 13 /product= “Xaa denotes Nle” 7 Gly Glu Gly Thr Phe ThrSer Asp Leu Ser Lys Gln Xaa Glu Glu Glu 1 5 10 15 Ala Val Arg Leu PheIle Glu Trp Leu Lys Asn Gly Arg 20 25 30 amino acids amino acid singlestrand linear peptide 8 His Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser LysGln Met Glu Glu 1 5 10 15 Glu Ala Val Arg Ala Phe Ile Glu Trp Leu LysAsn Gly Arg 20 25 30 30 amino acids amino acid single strand linearpeptide 9 His Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Met GluGlu 1 5 10 15 Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Lys Ala Gly Arg 2025 30 30 amino acids amino acid single strand linear peptide 10 His GlyGlu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Met Glu Glu 1 5 10 15 GluAla Val Arg Ala Phe Ile Glu Trp Leu Lys Ala Gly Arg 20 25 30 30 aminoacids amino acid single strand linear peptide misc_feature 30 /product=“-OH replaces terminal -NH2” 11 His Gly Glu Gly Thr Phe Thr Ser Asp LeuSer Lys Gln Ala Glu Glu 1 5 10 15 Glu Ala Val Arg Leu Phe Ile Glu TrpLeu Lys Asn Gly Arg 20 25 30 30 amino acids amino acid single strandlinear peptide misc_feature /product= “Modified by Ac” 12 His Gly GluGly Thr Phe Thr Ser Asp Leu Ser Lys Gln Ile Glu Glu 1 5 10 15 Glu AlaVal Arg Leu Phe Ile Glu Trp Leu Lys Asn Gly Arg 20 25 30 27 amino acidsamino acid single strand linear peptide peptide 14 /product= “Xaadenotes Nle” 13 His Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln XaaGlu Glu 1 5 10 15 Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Lys 20 25 30amino acids amino acid single strand linear peptide 14 His Ser Asp GlyThr Phe Thr Ser Asp Leu Ser Lys Gln Ala Glu Glu 1 5 10 15 Glu Ala ValArg Leu Phe Ile Glu Trp Leu Lys Asn Ala Arg 20 25 30 30 amino acidsamino acid single strand linear peptide 15 His Ser Asp Gly Thr Phe ThrSer Asp Leu Ser Lys Gln Ala Glu Glu 1 5 10 15 Glu Ala Val Arg Leu PheIle Glu Trp Leu Ala Asn Gly Arg 20 25 30 30 amino acids amino acidlinear peptide 16 His Ser Asp Gly Thr Phe Thr Ser Asp Leu Ser Lys GlnAla Glu Glu 1 5 10 15 Glu Ala Val Arg Leu Phe Ile Glu Trp Ala Lys AsnGly Arg 20 25 30 30 amino acids amino acid single strand linear peptidepeptide 14 /product= “Xaa denotes Nle” 17 His Ala Glu Gly Thr Phe ThrSer Asp Leu Ser Lys Gln Xaa Glu Glu 1 5 10 15 Glu Ala Val Arg Leu PheIle Glu Trp Leu Lys Asn Gly Arg 20 25 30 30 amino acids amino acidsingle strand linear peptide peptide 14 /product= “Xaa denotes Nle” 18His Cys Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Xaa Glu Glu 1 5 1015 Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Lys Asn Gly Arg 20 25 30 30amino acids amino acid single strand linear peptide peptide 14 /product=“Xaa denotes Nle” 19 His Asp Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys GlnXaa Glu Glu 1 5 10 15 Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Lys AsnGly Arg 20 25 30 30 amino acids amino acid single strand linear peptidepeptide 14 /product= “Xaa denotes Nle” 20 His Glu Glu Gly Thr Phe ThrSer Asp Leu Ser Lys Gln Xaa Glu Glu 1 5 10 15 Glu Ala Val Arg Leu PheIle Glu Trp Leu Lys Asn Gly Arg 20 25 30 30 amino acids amino acidsingle strand linear peptide peptide 14 /product= “Xaa denotes Nle” 21His Phe Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Xaa Glu Glu 1 5 1015 Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Lys Asn Gly Arg 20 25 30 30amino acids amino acid single strand linear peptide peptide 14 /product=“Xaa denotes Nle” 22 His His Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys GlnXaa Glu Glu 1 5 10 15 Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Lys AsnGly Arg 20 25 30 30 amino acids amino acid single strand linear peptidepeptide 14 /product= “Xaa denotes Nle” 23 His Ile Glu Gly Thr Phe ThrSer Asp Leu Ser Lys Gln Xaa Glu Glu 1 5 10 15 Glu Ala Val Arg Leu PheIle Glu Trp Leu Lys Asn Gly Arg 20 25 30 30 amino acids amino acidsingle strand linear peptide peptide 14 /product= “Xaa denotes Nle” 24His Lys Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Xaa Glu Glu 1 5 1015 Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Lys Asn Gly Arg 20 25 30 30amino acids amino acid single strand linear peptide peptide 14 /product=“Xaa denotes Nle” 25 His Leu Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys GlnXaa Glu Glu 1 5 10 15 Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Lys AsnGly Arg 20 25 30 30 amino acids amino acid single strand linear peptidepeptide 14 /product= “Xaa denotes Nle” 26 His Met Glu Gly Thr Phe ThrSer Asp Leu Ser Lys Gln Xaa Glu Glu 1 5 10 15 Glu Ala Val Arg Leu PheIle Glu Trp Leu Lys Asn Gly Arg 20 25 30 30 amino acids amino acidsingle strand linear peptide peptide 14 /product= “Xaa denotes Nle” 27His Asn Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Xaa Glu Glu 1 5 1015 Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Lys Asn Gly Arg 20 25 30 30amino acids amino acid single strand linear peptide peptide 14 /product=“Xaa denotes Nle” 28 His Pro Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys GlnXaa Glu Glu 1 5 10 15 Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Lys AsnGly Arg 20 25 30 30 amino acids amino acid single strand linear peptidepeptide 14 /product= “Xaa denotes Nle” 29 His Gln Glu Gly Thr Phe ThrSer Asp Leu Ser Lys Gln Xaa Glu Glu 1 5 10 15 Glu Ala Val Arg Leu PheIle Glu Trp Leu Lys Asn Gly Arg 20 25 30 30 amino acids amino acidsingle strand linear peptide peptide 14 /product= “Xaa denotes Nle” 30His Arg Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Xaa Glu Glu 1 5 1015 Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Lys Asn Gly Arg 20 25 30 30amino acids amino acid single strand linear peptide peptide 14 /product=“Xaa denotes Nle” 31 His Ser Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys GlnXaa Glu Glu 1 5 10 15 Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Lys AsnGly Arg 20 25 30 30 amino acids amino acid single strand linear peptidepeptide 14 /product= “Xaa denotes Nle” 32 His Thr Glu Gly Thr Phe ThrSer Asp Leu Ser Lys Gln Xaa Glu Glu 1 5 10 15 Glu Ala Val Arg Leu PheIle Glu Trp Leu Lys Asn Gly Arg 20 25 30 30 amino acids amino acidsingle strand linear peptide peptide 14 /product= “Xaa denotes Nle” 33His Val Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Xaa Glu Glu 1 5 1015 Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Lys Asn Gly Arg 20 25 30 30amino acids amino acid single strand linear peptide peptide 14 /product=“Xaa denotes Nle” 34 His Trp Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys GlnXaa Glu Glu 1 5 10 15 Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Lys AsnGly Arg 20 25 30 30 amino acids amino acid single strand linear peptidepeptide 14 /product= “Xaa denotes Nle” 35 His Tyr Glu Gly Thr Phe ThrSer Asp Leu Ser Lys Gln Xaa Glu Glu 1 5 10 15 Glu Ala Val Arg Leu PheIle Glu Trp Leu Lys Asn Gly Arg 20 25 30 30 amino acids amino acidsingle strand linear peptide 36 His Ala Asp Gly Thr Phe Thr Ser Asp LeuSer Ser Tyr Met Glu Gly 1 5 10 15 Gln Ala Val Lys Glu Phe Ile Ala TrpLeu Val Lys Gly Arg 20 25 30 30 amino acids amino acid single strandlinear peptide 37 His Ser Asp Gly Thr Phe Thr Ser Asp Leu Ser Lys GlnAla Glu Glu 1 5 10 15 Glu Ala Val Arg Leu Phe Ile Glu Ala Leu Lys AsnGly Arg 20 25 30 30 amino acids amino acid single strand linear peptide38 His Ser Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Ala Glu Glu 1 510 15 Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Lys Asn Gly Arg 20 25 3030 amino acids amino acid single strand linear peptide 39 Ala Ser AspGly Thr Phe Thr Ser Asp Leu Ser Lys Gln Val Glu Glu 1 5 10 15 Glu AlaVal Arg Leu Phe Ile Glu Trp Leu Lys Asn Gly Arg 20 25 30 30 amino acidsamino acid single strand linear peptide 40 His Gly Ala Gly Thr Phe ThrSer Asp Leu Ser Lys Gln Ala Glu Glu 1 5 10 15 Glu Ala Val Arg Leu PheIle Glu Trp Leu Lys Asn Gly Arg 20 25 30 30 amino acids amino acidsingle strand linear peptide 41 His Gly Glu Gly Ala Phe Thr Ser Asp LeuSer Lys Gln Ala Glu Glu 1 5 10 15 Glu Ala Val Arg Leu Phe Ile Glu TrpLeu Lys Asn Gly Arg 20 25 30 30 amino acids amino acid single strandlinear peptide 42 His Gly Glu Gly Tyr Phe Thr Ser Asp Leu Ser Lys GlnAla Glu Glu 1 5 10 15 Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Lys AsnGly Arg 20 25 30 30 amino acids amino acid single strand linear peptide43 His Gly Glu Gly Thr Tyr Thr Ser Asp Leu Ser Lys Gln Ala Glu Glu 1 510 15 Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Lys Asn Gly Arg 20 25 3030 amino acids amino acid single strand linear peptide 44 His Gly GluGly Thr Ile Thr Ser Asp Leu Ser Lys Gln Ala Glu Glu 1 5 10 15 Glu AlaVal Arg Leu Phe Ile Glu Trp Leu Lys Asn Gly Arg 20 25 30 30 amino acidsamino acid single strand linear peptide 45 His Gly Glu Gly Thr Phe SerSer Asp Leu Ser Lys Gln Ala Glu Glu 1 5 10 15 Glu Ala Val Arg Leu PheIle Glu Trp Leu Lys Asn Gly Arg 20 25 30 30 amino acids amino acidsingle strand linear peptide 46 His Gly Glu Gly Thr Phe Tyr Ser Asp LeuSer Lys Gln Ala Glu Glu 1 5 10 15 Glu Ala Val Arg Leu Phe Ile Glu TrpLeu Lys Asn Gly Arg 20 25 30 30 amino acids amino acid single strandlinear peptide 47 His Gly Glu Gly Thr Phe Thr Thr Asp Leu Ser Lys GlnAla Glu Glu 1 5 10 15 Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Lys AsnGly Arg 20 25 30 30 amino acids amino acid single strand linear peptide48 His Gly Glu Gly Thr Phe Thr Tyr Asp Leu Ser Lys Gln Ala Glu Glu 1 510 15 Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Lys Asn Gly Arg 20 25 3030 amino acids amino acid single strand linear peptide 49 His Gly GluGly Thr Phe Thr Ser Glu Leu Ser Lys Gln Ala Glu Glu 1 5 10 15 Glu AlaVal Arg Leu Phe Ile Glu Trp Leu Lys Asn Gly Arg 20 25 30 30 amino acidsamino acid single strand linear peptide 50 His Gly Glu Gly Thr Phe ThrSer Asp Ala Ser Lys Gln Ala Glu Glu 1 5 10 15 Glu Ala Val Arg Leu PheIle Glu Trp Leu Lys Asn Gly Arg 20 25 30 30 amino acids amino acidsingle strand linear peptide 51 His Gly Glu Gly Thr Phe Thr Ser Asp LeuAla Lys Gln Ala Glu Glu 1 5 10 15 Glu Ala Val Arg Leu Phe Ile Glu TrpLeu Lys Asn Gly Arg 20 25 30 30 amino acids amino acid single strandlinear peptide 52 His Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser Ala GlnAla Glu Glu 1 5 10 15 Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Lys AsnGly Arg 20 25 30 30 amino acids amino acid single strand linear peptide53 His Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Ala Ala Glu Glu 1 510 15 Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Lys Asn Gly Arg 20 25 3030 amino acids amino acid single strand linear peptide 54 His Gly GluGly Thr Phe Thr Ser Asp Leu Ser Lys Gln Ala Ala Glu 1 5 10 15 Glu AlaVal Arg Leu Phe Ile Glu Trp Leu Lys Asn Gly Arg 20 25 30 30 amino acidsamino acid single strand linear peptide 55 Gly Ser Asp Gly Ser Phe ThrSer Asp Leu Ser Lys Gln Ala Glu Ala 1 5 10 15 Glu Ala Val Arg Leu PheIle Glu Trp Leu Lys Asn Gly Arg 20 25 30 30 amino acids amino acidsingle strand linear peptide 56 Lys Gly Glu Gly Thr Phe Thr Ser Asp LeuSer Lys Gln Ala Glu Glu 1 5 10 15 Ala Ala Val Arg Leu Phe Ile Glu TrpLeu Lys Asn Gly Arg 20 25 30 30 amino acids amino acid single strandlinear peptide 57 His Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys GlnAla Glu Glu 1 5 10 15 Glu Leu Val Arg Leu Phe Ile Glu Trp Leu Lys AsnGly Arg 20 25 30 30 amino acids amino acid single strand linear peptide58 His Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Ala Glu Glu 1 510 15 Glu Ala Ile Arg Leu Phe Ile Glu Trp Leu Lys Asn Gly Arg 20 25 3030 amino acids amino acid single strand linear peptide 59 His Gly GluGly Thr Phe Thr Ser Asp Leu Ser Lys Gln Ala Glu Glu 1 5 10 15 Glu AlaVal Ala Leu Phe Ile Glu Trp Leu Lys Asn Gly Arg 20 25 30 30 amino acidsamino acid single strand linear peptide 60 His Ser Asp Gly Thr Phe ThrSer Asp Leu Ser Lys Gln Ala Glu Glu 1 5 10 15 Glu Ala Val Arg Leu TyrIle Glu Trp Leu Lys Asn Gly Arg 20 25 30 30 amino acids amino acidsingle strand linear peptide 61 His Gly Glu Gly Thr Phe Thr Ser Asp LeuSer Lys Gln Ala Glu Glu 1 5 10 15 Glu Ala Val Arg Leu Phe Val Glu TrpLeu Lys Asn Gly Arg 20 25 30 30 amino acids amino acid single strandlinear peptide 62 His Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys GlnAla Glu Glu 1 5 10 15 Glu Ala Val Arg Leu Phe Ile Leu Trp Leu Lys AsnGly Arg 20 25 30 30 amino acids amino acid single strand linear peptide63 His Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Ala Glu Glu 1 510 15 Glu Ala Val Arg Leu Phe Ile Glu Ala Leu Lys Asn Gly Arg 20 25 3030 amino acids amino acid single strand linear peptide 64 His Gly GluGly Thr Phe Thr Ser Asp Leu Ser Lys Gln Ala Glu Glu 1 5 10 15 Glu AlaVal Arg Leu Phe Ile Glu Trp Ala Lys Asn Gly Arg 20 25 30 30 amino acidsamino acid single strand linear peptide 65 His Gly Glu Gly Thr Phe ThrSer Asp Leu Ser Lys Gln Ala Glu Glu 1 5 10 15 Glu Ala Val Arg Leu PheIle Glu Trp Leu Ala Asn Gly Arg 20 25 30 30 amino acids amino acidsingle strand linear peptide 66 His Gly Glu Gly Thr Phe Thr Ser Asp LeuSer Lys Gln Ala Glu Glu 1 5 10 15 Glu Ala Val Arg Leu Phe Ile Glu TrpLeu Lys Asn Ala Arg 20 25 30 30 amino acids amino acid single strandlinear peptide 67 His Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys GlnAla Glu Glu 1 5 10 15 Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Lys AsnGly Arg 20 25 30 30 amino acids amino acid single strand linear peptide68 His Ser Asp Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Met Glu Glu 1 510 15 Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Lys Asn Gly Arg 20 25 3030 amino acids amino acid single strand linear peptide peptide 14/product= “Xaa denotes Nle” 69 His Ser Asp Gly Thr Phe Thr Ser Asp LeuSer Lys Gln Xaa Glu Glu 1 5 10 15 Glu Ala Val Arg Leu Phe Ile Glu TrpLeu Lys Asn Gly Tyr 20 25 30 30 amino acids amino acid single strandlinear peptide peptide 14 /product= “Xaa denotes Nle” misc_feature 30/product= “-OH replaces terminal -NH2” 70 His Ser Asp Gly Thr Phe ThrSer Asp Leu Ser Lys Gln Xaa Glu Glu 1 5 10 15 Glu Ala Val Arg Leu PheIle Glu Trp Leu Lys Asn Gly Arg 20 25 30 30 amino acids amino acidsingle strand linear peptide 71 His Ser Asp Gly Thr Phe Thr Ser Asp LeuSer Lys Gln Ala Glu Glu 1 5 10 15 Glu Ala Val Arg Leu Phe Ile Glu TrpLeu Lys Asn Gly Arg 20 25 30 29 amino acids amino acid single strandlinear peptide peptide 13 /product= “Xaa denotes Nle” 72 Ser Asp Gly ThrPhe Thr Ser Asp Leu Ser Lys Gln Xaa Glu Glu Glu 1 5 10 15 Ala Val ArgLeu Phe Ile Glu Trp Leu Lys Asn Gly Arg 20 25 28 amino acids amino acidsingle strand linear peptide peptide 12 /product= “Xaa denotes Nle” 73Asp Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Xaa Glu Glu Glu Ala 1 5 1015 Val Arg Leu Phe Ile Glu Trp Leu Lys Asn Gly Arg 20 25 29 amino acidsamino acid single strand linear peptide misc_feature /product= “Modifiedby Ac” peptide 13 /product= “Xaa denotes Nle” 74 Ser Asp Gly Thr Phe ThrSer Asp Leu Ser Lys Gln Xaa Glu Glu Glu 1 5 10 15 Ala Val Arg Leu PheIle Glu Trp Leu Lys Asn Gly Arg 20 25 28 amino acids amino acid singlestrand linear peptide misc_feature /product= “Modified by Ac” peptide 12/product= “Xaa denotes Nle” 75 Asp Gly Thr Phe Thr Ser Asp Leu Ser LysGln Xaa Glu Glu Glu Ala 1 5 10 15 Val Arg Leu Phe Ile Glu Trp Leu LysAsn Gly Arg 20 25 27 amino acids amino acid single strand linear peptidepeptide 14 /product= “Xaa denotes Nle” 76 His Ser Asp Gly Thr Phe ThrSer Asp Leu Ser Lys Gln Xaa Glu Glu 1 5 10 15 Glu Ala Val Arg Leu PheIle Glu Trp Leu Lys 20 25 30 amino acids amino acid single strand linearpeptide 77 His Lys Pro Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Ala GluGlu 1 5 10 15 Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Lys Asn Gly Arg 2025 30 30 amino acids amino acid single strand linear peptide peptide 14/product= “Xaa denotes Nle” 78 His Ala Asp Gly Thr Phe Thr Ser Asp LeuSer Lys Gln Xaa Glu Glu 1 5 10 15 Glu Ala Val Arg Leu Phe Ile Glu TrpLeu Lys Asn Gly Arg 20 25 30 30 amino acids amino acid single strandlinear peptide peptide 14 /product= “Xaa denotes Nle” 79 His Cys Asp GlyThr Phe Thr Ser Asp Leu Ser Lys Gln Xaa Glu Glu 1 5 10 15 Glu Ala ValArg Leu Phe Ile Glu Trp Leu Lys Asn Gly Arg 20 25 30 30 amino acidsamino acid single strand linear peptide peptide 14 /product= “Xaadenotes Nle” 80 His Asp Asp Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln XaaGlu Glu 1 5 10 15 Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Lys Asn GlyArg 20 25 30 30 amino acids amino acid single strand linear peptidepeptide 14 /product= “Xaa denotes Nle” 81 His Glu Asp Gly Thr Phe ThrSer Asp Leu Ser Lys Gln Xaa Glu Glu 1 5 10 15 Glu Ala Val Arg Leu PheIle Glu Trp Leu Lys Asn Gly Arg 20 25 30 30 amino acids amino acidsingle strand linear peptide peptide 14 /product= “Xaa denotes Nle” 82His Phe Asp Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Xaa Glu Glu 1 5 1015 Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Lys Asn Gly Arg 20 25 30 30amino acids amino acid single strand linear peptide peptide 14 /product=“Xaa denotes Nle” 83 His Gly Asp Gly Thr Phe Thr Ser Asp Leu Ser Lys GlnXaa Glu Glu 1 5 10 15 Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Lys AsnGly Arg 20 25 30 30 amino acids amino acid single strand linear peptidepeptide 14 /product= “Xaa denotes Nle” 84 His His Asp Gly Thr Phe ThrSer Asp Leu Ser Lys Gln Xaa Glu Glu 1 5 10 15 Glu Ala Val Arg Leu PheIle Glu Trp Leu Lys Asn Gly Arg 20 25 30 30 amino acids amino acidsingle strand linear peptide peptide 14 /product= “Xaa denotes Nle” 85His Ile Asp Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Xaa Glu Glu 1 5 1015 Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Lys Asn Gly Arg 20 25 30 30amino acids amino acid single strand linear peptide peptide 14 /product=“Xaa denotes Nle” 86 His Lys Asp Gly Thr Phe Thr Ser Asp Leu Ser Lys GlnXaa Glu Glu 1 5 10 15 Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Lys AsnGly Arg 20 25 30 30 amino acids amino acid single strand linear peptidepeptide 14 /product= “Xaa denotes Nle” 87 His Leu Asp Gly Thr Phe ThrSer Asp Leu Ser Lys Gln Xaa Glu Glu 1 5 10 15 Glu Ala Val Arg Leu PheIle Glu Trp Leu Lys Asn Gly Arg 20 25 30 30 amino acids amino acidsingle strand linear peptide peptide 14 /product= “Xaa denotes Nle” 88His Met Asp Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Xaa Glu Glu 1 5 1015 Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Lys Asn Gly Arg 20 25 30 30amino acids amino acid single strand linear peptide peptide 14 /product=“Xaa denotes Nle” 89 His Asn Asp Gly Thr Phe Thr Ser Asp Leu Ser Lys GlnXaa Glu Glu 1 5 10 15 Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Lys AsnGly Arg 20 25 30 30 amino acids amino acid single strand linear peptidepeptide 14 /product= “Xaa denotes Nle” 90 His Pro Asp Gly Thr Phe ThrSer Asp Leu Ser Lys Gln Xaa Glu Glu 1 5 10 15 Glu Ala Val Arg Leu PheIle Glu Trp Leu Lys Asn Gly Arg 20 25 30 30 amino acids amino acidsingle strand linear peptide peptide 14 /product= “Xaa denotes Nle” 91His Gln Asp Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Xaa Glu Glu 1 5 1015 Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Lys Asn Gly Arg 20 25 30 30amino acids amino acid single strand linear peptide peptide 14 /product=“Xaa denotes Nle” 92 His Arg Asp Gly Thr Phe Thr Ser Asp Leu Ser Lys GlnXaa Glu Glu 1 5 10 15 Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Lys AsnGly Arg 20 25 30 30 amino acids amino acid single strand linear peptidepeptide 14 /product= “Xaa denotes Nle” 93 His Thr Asp Gly Thr Phe ThrSer Asp Leu Ser Lys Gln Xaa Glu Glu 1 5 10 15 Glu Ala Val Arg Leu PheIle Glu Trp Leu Lys Asn Gly Arg 20 25 30 30 amino acids amino acidsingle strand linear peptide peptide 14 /product= “Xaa denotes Nle” 94His Val Asp Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Xaa Glu Glu 1 5 1015 Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Lys Asn Gly Arg 20 25 30 30amino acids amino acid single strand linear peptide peptide 14 /product=“Xaa denotes Nle” 95 His Trp Asp Gly Thr Phe Thr Ser Asp Leu Ser Lys GlnXaa Glu Glu 1 5 10 15 Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Lys AsnGly Arg 20 25 30 30 amino acids amino acid single strand linear peptidepeptide 14 /product= “Xaa denotes Nle” 96 His Tyr Asp Gly Thr Phe ThrSer Asp Leu Ser Lys Gln Xaa Glu Glu 1 5 10 15 Glu Ala Val Arg Leu PheIle Glu Trp Leu Lys Asn Gly Arg 20 25 30 30 amino acids amino acidsingle strand linear peptide 97 His Ser Ala Gly Thr Phe Thr Ser Asp LeuSer Lys Gln Ala Glu Glu 1 5 10 15 Glu Ala Val Arg Leu Phe Ile Glu TrpLeu Lys Asn Gly Arg 20 25 30 30 amino acids amino acid single strandlinear peptide 98 His Ser Asp Gly Ala Phe Thr Ser Asp Leu Ser Lys GlnAla Glu Glu 1 5 10 15 Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Lys AsnGly Arg 20 25 30 30 amino acids amino acid single strand linear peptide99 His Ser Asp Gly Tyr Phe Thr Ser Asp Leu Ser Lys Gln Ala Glu Glu 1 510 15 Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Lys Asn Gly Arg 20 25 3030 amino acids amino acid single strand linear peptide 100 His Ser AspGly Thr Tyr Thr Ser Asp Leu Ser Lys Gln Ala Glu Glu 1 5 10 15 Glu AlaVal Arg Leu Phe Ile Glu Trp Leu Lys Asn Gly Arg 20 25 30 30 amino acidsamino acid single strand linear peptide 101 His Ser Asp Gly Thr Ile ThrSer Asp Leu Ser Lys Gln Ala Glu Glu 1 5 10 15 Glu Ala Val Arg Leu PheIle Glu Trp Leu Lys Asn Gly Arg 20 25 30 30 amino acids amino acidsingle strand linear peptide 102 His Ser Asp Gly Thr Phe Ser Ser Asp LeuSer Lys Gln Ala Glu Glu 1 5 10 15 Glu Ala Val Arg Leu Phe Ile Glu TrpLeu Lys Asn Gly Arg 20 25 30 30 amino acids amino acid single strandlinear peptide 103 His Ser Asp Gly Thr Phe Tyr Ser Asp Leu Ser Lys GlnAla Glu Glu 1 5 10 15 Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Lys AsnGly Arg 20 25 30 30 amino acids amino acid single strand linear peptide104 His Ser Asp Gly Thr Phe Thr Thr Asp Leu Ser Lys Gln Ala Glu Glu 1 510 15 Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Lys Asn Gly Arg 20 25 3030 amino acids amino acid single strand linear peptide 105 His Ser AspGly Thr Phe Thr Tyr Asp Leu Ser Lys Gln Ala Glu Glu 1 5 10 15 Glu AlaVal Arg Leu Phe Ile Glu Trp Leu Lys Asn Gly Arg 20 25 30 30 amino acidsamino acid single strand linear peptide 106 His Ser Asp Gly Thr Phe ThrSer Glu Leu Ser Lys Gln Ala Glu Glu 1 5 10 15 Glu Ala Val Arg Leu PheIle Glu Trp Leu Lys Asn Gly Arg 20 25 30 30 amino acids amino acidsingle strand linear peptide 107 His Ser Asp Gly Thr Phe Thr Ser Asp AlaSer Lys Gln Ala Glu Glu 1 5 10 15 Glu Ala Val Arg Leu Phe Ile Glu TrpLeu Lys Asn Gly Arg 20 25 30 30 amino acids amino acid single strandlinear peptide 108 His Ser Asp Gly Thr Phe Thr Ser Asp Leu Ala Lys GlnAla Glu Glu 1 5 10 15 Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Lys AsnGly Arg 20 25 30 30 amino acids amino acid single strand linear peptide109 His Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser Ala Gln Ala Glu Glu 1 510 15 Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Lys Asn Gly Arg 20 25 3030 amino acids amino acid single strand linear peptide 110 His Ser AspGly Thr Phe Thr Ser Asp Leu Ser Lys Ala Ala Glu Glu 1 5 10 15 Glu AlaVal Arg Leu Phe Ile Glu Trp Leu Lys Asn Gly Arg 20 25 30 30 amino acidsamino acid single strand linear peptide 111 His Ser Asp Gly Thr Phe ThrSer Asp Leu Ser Lys Gln Ala Ala Glu 1 5 10 15 Glu Ala Val Arg Leu PheIle Glu Trp Leu Lys Asn Gly Arg 20 25 30 30 amino acids amino acidsingle strand linear peptide 112 Lys Ser Asp Gly Thr Phe Thr Ser Asp LeuSer Lys Gln Ala Glu Glu 1 5 10 15 Ala Ala Val Arg Leu Phe Ile Glu TrpLeu Lys Asn Gly Arg 20 25 30 30 amino acids amino acid single strandlinear peptide 113 His Ser Asp Gly Thr Phe Thr Ser Asp Leu Ser Lys GlnAla Glu Glu 1 5 10 15 Glu Leu Val Arg Leu Phe Ile Glu Trp Leu Lys AsnGly Arg 20 25 30 30 amino acids amino acid single strand linear peptide114 His Ser Asp Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Ala Glu Glu 1 510 15 Glu Ala Ile Arg Leu Phe Ile Glu Trp Leu Lys Asn Gly Arg 20 25 3030 amino acids amino acid single strand linear peptide 115 His Ser AspGly Thr Phe Thr Ser Asp Leu Ser Lys Gln Ala Glu Glu 1 5 10 15 Glu AlaVal Ala Leu Phe Ile Glu Trp Leu Lys Asn Gly Arg 20 25 30 30 amino acidsamino acid single strand linear peptide 116 His Ser Asp Gly Thr Phe ThrSer Asp Leu Ser Lys Gln Ala Glu Glu 1 5 10 15 Glu Ala Val Arg Leu TyrIle Glu Trp Leu Lys Asn Gly Arg 20 25 30 30 amino acids amino acidsingle strand linear peptide 117 His Ser Asp Gly Thr Phe Thr Ser Asp LeuSer Lys Gln Ala Glu Glu 1 5 10 15 Glu Ala Val Arg Leu Phe Val Glu TrpLeu Lys Asn Gly Arg 20 25 30 30 amino acids amino acid single strandlinear peptide 118 His Ser Asp Gly Thr Phe Thr Ser Asp Leu Ser Lys GlnAla Glu Glu 1 5 10 15 Glu Ala Val Arg Leu Phe Ile Leu Trp Leu Lys AsnGly Arg 20 25 30 28 amino acids amino acid single strand linear peptidemisc_feature /product= “Modified by Suc” peptide 12 /product= “Xaadenotes Nle” 119 Asp Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Xaa Glu GluGlu Ala 1 5 10 15 Val Arg Leu Phe Ile Glu Trp Leu Lys Asn Gly Arg 20 2530 amino acids amino acid single strand linear peptide peptide 14/product= “Xaa denotes Nle” 120 His Ser Asp Gly Thr Phe Thr Ser Asp LeuSer Lys Gln Xaa Glu Glu 1 5 10 15 Glu Ala Val Arg Leu Phe Ile Glu TrpLeu Lys Asn Gly Arg 20 25 30

What is claimed is:
 1. A peptide consisting essentially of SEQ ID NO:1or SEQ ID NO:2 SEQ ID NO:1 1               5                   10 HisSer Asp Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln    15                  20                  25 Met Glu Glu Glu Ala ValArg Leu Phe Ile Glu Trp Leu             30 Lys Asn Gly Xaa SEQ ID NO:21               5                   10 His Gly Glu Gly Thr Phe Thr SerAsp Leu Ser Lys Gln     15                  20                  25 MetGlu Glu Glu Ala Val Arg Leu Phe Ile Glu Trp Leu             30 Lys AsnGly Xaa

in which Xaa specifies one of the proteinogenic amino acids: Asp, Glu,Arg, His, Lys, Ala, Asn, Cys, Gln, Ile, Leu, Met, Phe, Pro, Ser, Thr,Trp, Tyr, Val or a non-proteinogenic amino acid, and wherein inpositions 1, 2, 28, 29 or 30, independent of one another, amino acidsare optionally part of the sequence, and the N-terminus is representedby NR₁R₂ in which R₁ denotes hydrogen, acetyl, trifluoroacetyl,adamantyl, Fmoc, benzyloxycarbonyl, Boc, Alloc, C₁-C₆ alkyl, C₂-C₈alkenyl or C₇-C₉ aralkyl, R₂ denotes hydrogen, acetyl, trifluoroacetyl,adamantyl, Fmoc, benzyloxycarbonyl, Boc, Alloc, C₁-C₆ alkyl, C₂-C₈alkenyl or C₇-C₉ aralkyl, and the C-terminus is represented by COR₃ inwhich R₃ equals OR₄ or NR₄R₅ in which R₄ equals hydrogen or C₁-C₆ alkylin which R₅ equals hydrogen or C₁-C₆ alkyl, as well as physiologicallytolerated salts and esters of the foregoing.
 2. A peptide as claimed inclaim 1, wherein the Xaa amino acid at position 30 in SEQ ID NO:1 or SEQID NO:2 is Arg, D-Arg, Tyr or D-Tyr.
 3. A peptide as claimed in claim 2,wherein the Xaa amino acid at position 30 in SEQ ID NO:1 or SEQ ID NO:2is Arg or Tyr.
 4. A peptide as claimed in claim 1, in which Xaaspecifies one of the non-proteinogenic amino acids amino acids Dab, Dap,Om, β-Ala, Aib, Cit, Cha, Oak, Mab, Pab, Amb, Ahx, Ahp, Aoc, Ade, Atd,Nal, Nle, Sar, Tic.
 5. A peptide consisting essentially of SEQ ID NO:1or SEQ ID NO:2 wherein the peptide has one of the sequences identifiedby sequence ID NOS. 5, 68, 69, 71, 78-82 or 84-91, and wherein theN-terminus is represented by NR₁R₂ in which R₁ denotes hydrogen, acetyl,trifluoroacetyl, adamantyl, Fmoc, benzyloxycarbonyl, Boc, Alloc, C₁-C₆alkyl, C₂-C₈ alkenyl or C₇-C₉ aralkyl, R₂ denotes hydrogen, acetyl,trifluoroacetyl, adamantyl, Fmoc, benzyloxycarbonyl, Boc, Alloc, C₁-C₆alkyl, C₂-C₈ alkenyl or C₇-C₉ aralkyl, and the C-terminus is representedby COR₃ in which R₃ equals OR₄ or NR₄R₅ in which R₄ equals hydrogen orC₁-C₆ alkyl in which R₅ equals hydrogen or C₁-C₆ alkyl as well asphysiologically tolerated salts and esters thereof.
 6. A pharmaceuticalpreparation for stimulating the release of insulin comprising at leastone peptide as claimed in claim 1, in addition to physiologicallyacceptable carriers and auxiliary substances.
 7. A pharmaceuticalcomposition comprising peptides as claimed in claim 1 for the treatmentof diabetes.
 8. A method of stimulating the release of insulin in amammal comprising administering to the mammal a peptide according toclaim 1 in an amount sufficient to release insulin.
 9. The method ofclaim 8, wherein the mammal is a human.
 10. A peptide consistingessentially of SEQ ID NO:1 or SEQ ID NO:2 SEQ ID NO:11               5                   10 His Ser Asp Gly Thr Phe Thr SerAsp Leu Ser Lys Gln     15                  20                  25 MetGlu Glu Glu Ala Val Arg Leu Phe Ile Glu Trp Leu             30 Lys AsnGly Xaa SEQ ID NO:2 1               5                   10 His Gly GluGly Thr Phe Thr Ser Asp Leu Ser Lys Gln    15                  20                  25 Met Glu Glu Glu Ala ValArg Leu Phe Ile Glu Trp Leu             30 Lys Asn Glu Xaa

in which Xaa specifies one of the proteinogenic amino acids: Asp, Glu,Arg, His, Lys, Ala, Asn, Cys, Gln, Ile, Leu, Met, Phe, Pro, Ser, Thr,Trp, Tyr, Val or a non-proteinogenic amino acid, and wherein inpositions 1, 2, 28, 29 or 30, independent of one another, amino acidsare optionally part of the sequence, and the N-terminus is representedby NR₁R₂ in which R₁ denotes hydrogen, acetyl, trifluoroacetyl,adamantyl, Fmoc, benzyloxycarbonyl Boc, Alloc, C₁-C₆ alkyl, C₂-C₈alkenyl or C₇-C₉ aralkyl, R₂ denotes hydrogen, acetyl, trifluoroacetyl,adamantyl, Fmoc, benzyloxycarbonyl, Boc, Alloc, C₁-C₆ alkyl, C₂-C₈alkenyl or C₇-C₉ aralkyl, and the C-terminus is represented by COR₃ inwhich R₃ equals OR₄ or NR₄R₅ in which R₄ equals hydrogen or C₁-C₆ alkylin which R₅ equals hydrogen or C₁-C₆ alkyl as well as physiologicallytolerated salts and esters thereof, with the proviso that at least onebut at most 10 of the following modifications (a) to (o) apply to theamino acid chain: (a) the α-amino acid in position 1 is D-His, Ala,D-Ala, Gly, Lys or D-Lys; (b) the α-amino acid in position 2 is Ser,D-Ser, Thr, D-Thr, Ala, D-Ala, Ile, D-Ile, Val, D-Val, Leu, or D-Leu,Cys, Asp, Glu, Phe, His, Lys, Met, Asn, Pro, Gln, Arg, Trp or Tyr; (c)the α-amino acid in position 3 is D-Glu, D-Asp, Ala or D-Ala; (d) theamino acid in position 4 is Ala, D-Ala or β-Ala; (e) the α-amino acid inposition 5 is Ser, Tyr or Ala; (f) the α-amino acid in position 6 isAla, Ile, Val, Leu, Cha or Tyr; (g) the α-amino acid in position 7 isAla, D-Ala, Tyr, D-Tyr, Ser, D-Ser or D-Thr; (h) the α-amino acid inposition 8 is Ala, Tyr or Thr; (i) the α-amino acid in position 9 isAla, D-Ala, Glu, D-Glu or D-Asp; (j) the amino acids in positions 10,11, 12, 15, 16, 17, 18, 19, 20, 21, 24, 28, 29 are, independent of oneanother, one of the above-specified proteinogenic amino acids which aredifferent from the amino acid already present in the respectiveposition(s) in SEQ ID NO:1 or SEQ ID NO:2 or a non-proteinogenic D- orL-amino acid; (k) the α-amino acid in position 13 is a neutral L-aminoacid except Gln: (l) the α-amino acid in position 14 is replaced by aneutral L- or D-amino acid, except Met or L-leucine; (m) the α-aminoacid in position 22 is D-Phe, Tyr, D-Tyr, Leu, D-Leu, Val, D-Val, L-Cha,D-Cha, β-1-Nal, β-2-Nal or β-1-D-Nal; (n) the α-amino acid in position23 is Leu, D-Leu, D-Ile, Val, D-Val, L-Cha, D-Cha, Tyr, D-Tyr, Phe orD-Phe; and (o) the α-amino acid in positions 25, 26 or 27 is a neutralL- or D-amino acid, except Trp for position 25, Leu for position 26 andLys for position
 27. 11. A peptide as claimed in claim 10, wherein inSEQ ID NO:1 or SEQ ID NO:2 the α-amino acid in position 1 is Ala, Gly orLys.
 12. A peptide as claimed in claim 10, wherein in SEQ ID NO:1 or SEQID NO:2 the α-amino acid in position 2 is Thr, Ala, Val, Ile or Leu. 13.A peptide as claimed in claim 10, wherein in SEQ ID NO:1 or SEQ ID NO:2the α-amino acid in position 3 is Ala.
 14. A peptide as claimed in claim10, wherein in SEQ ID NO:1 or SEQ ID NO:2 the α-amino acid in position 4is Ala.
 15. A peptide as claimed in claim 10 wherein in SEQ ID NO:1 orSEQ ID NO:2 the α-amino acid in position 6 is Ala, Ile, Val, Leu or Tyr.16. A peptide as claimed in claim 10, wherein in SEQ ID NO:1 or SEQ IDNO:2 the α-amino acid in position 7 is Ala, Tyr or Ser.
 17. A peptide asclaimed in claim 10, wherein in SEQ ID NO:1 or SEQ ID NO:2 the α-aminoacid in position 9 is Ala or Glu.
 18. A peptide as claimed in claim 10wherein in SEQ ID NO:1 or SEQ ID NO:2 the α-amino acid in positions 10,11, 12, 15, 16, 17, 18, 19, 20, 21, 24, 28, 29 is a proteinogenicL-amino acid.
 19. A peptide as claimed in claim 10, wherein in SEQ IDNO:1 or SEQ ID NO:2 the α-amino acid in position 13 is a neutralproteinogenic L-amino acid.
 20. A peptide as claimed in claim 10,wherein in SEQ ID NO:1 or SEQ ID NO:2 the α-amino acid in position 14 isNle, D-Nle, Ala, D-Ala, Ile, D-Ile, Val or D-Val.
 21. A peptide asclaimed in claim 10, wherein in SEQ ID NO:1 or SEQ ID NO:2 the α-aminoacid in position 14 is Ile, Val or Ala.
 22. A peptide as claimed inclaim 10, wherein in SEQ ID NO:1 or SEQ ID NO:2 the α-amino acid inposition 22 is Tyr, Leu or Val.
 23. A peptide as claimed in claim 10,wherein in SEQ ID NO:1 or SEQ ID NO:2 the α-amino acid in position 23 isLeu, Val, Tyr or Phe.
 24. A peptide as claimed in claim 10, wherein inSEQ ID NO:1 or SEQ ID NO:2 the α-amino acid in positions 25, 26 or 27 isa proteinogenic L-amino acid.
 25. Peptide as claimed in claim 10,wherein in SEQ ID NO:1 or SEQ ID NO:2 an amino acid has been substitutedat position
 2. 26. Peptide as claimed in claim 10, wherein in SEQ IDNO:1 or SEQ ID NO:2 an amino acid has been substituted at position 14.27. Peptide as claimed in claim 10, wherein in SEQ ID NO:1 or SEQ IDNO:2 an amino acid has been substituted at position
 3. 28. A peptide of25-30 amino acids in length having at least 25 contiguous amino acids ofSEQ ID NO:1 or SEQ ID NO:2 SEQ ID NO:11               5                   10 His Gly Glu Gly Thr Phe Thr SerAsp Leu Ser Lys Gln     15                  20                  25 MetGlu Glu Glu Ala Val Arg Leu Phe Ile Glu Trp Leu             30 Lys AsnGlu Xaa SEQ ID NO:2 1               5                   10 His Gly GluGly Thr Phe Thr Ser Asp Leu Ser Lys Gln    15                  20                  25 Met Glu Glu Glu Ala ValArg Leu Phe Ile Glu Trp Leu             30 Lys Asn Glu Xaa

in which Xaa specifies one of the proteinogenic amino acids: Asp, Glu,Arg, His, Lys, Ala, Asn, Cys, Gln, Ile, Leu, Met, Phe, Pro, Ser, Thr,Trp, Tyr, Val or a non-proteinogenic amino acid, and wherein inpositions 1, 2, 28, 29 or 30, independent of one another, amino acidsare optionally part of the sequence, and the N-terminus is representedby NR₁R₂ in which R₁ denotes hydrogen, acetyl, trifluoroacetyl,adamantyl, Fmoc, benzyloxycarbonyl, Boc, Alloc, C₁-C₆ alkyl, C₂-C₈alkenyl or C₇-C₉ aralkyl, R₂ denotes hydrogen, acetyl, trifluoroacetyl,adamantyl, Fmoc, benzyloxycarbonyl, Boc, Alloc, C₁-C₆ alkyl, C₂-C₈alkenyl or C₇-C₉ aralkyl, and the C-terminus is represented by COR₃ inwhich R₃ equals OR₄ or NR₄R₅ in which R₄ equals hydrogen or C₁-C₆ alkylin which R₅ equals hydrogen or C₁-C₆ alkyl, as well as physiologicallytolerated salts and esters of the foregoing.
 29. A peptide as claimed inclaim 28, wherein the α amino acid at position 30 in SEQ ID NO:1 or SEQID NO:2 is Arg, D-Arg, Tyr or D-Tyr.
 30. A peptide as claimed in claim28, in which Xaa specifies one of the non-proteinogenic amino acidsamino acids Dab, Dap, Om, β-Ala, Aib, Cit, Cha, Oak, Mab, Pab, Amb, Ahx,Ahp, Aoc, Ade, Atd, Nal, Nle, Sar, Tic.
 31. Pharmaceutical preparationfor stimulating the release of insulin comprising at least one peptideas claimed in claim 28, in addition to physiologically acceptablecarriers and auxiliary substances.
 32. A pharmaceutical compositioncomprising peptides as claimed in claim 28 for the treatment ofdiabetes.
 33. A peptide of 25-30 amino acids in length wherein thepeptide has one of the sequences identified by SEQ ID NO:'S 5, 68, 69,71, 78-82 or 84-91, and wherein the N-terminus is represented by NR₁R₂in which R₁ denotes hydrogen, acetyl, trifluoroacetyl, adamantyl, Fmoc,benzyloxycarbonyl, Boc, Alloc, C₁-C₆ alkyl, C₂-C₈ alkenyl or C₇-C₉aralkyl, R₂ denotes hydrogen, acetyl, trifluoroacetyl, adamantyl, Fmoc,benzyloxycarbonyl, Boc, Alloc, C₁-C₆ alkyl, C₂-C₈ alkenyl or C₇-C₉aralkyl, and the C-terminus is represented by COR₃ in which R₃ equalsOR₄ or NR₄R₅ in which R₄ equals hydrogen or C₁-C₆ alkyl in which R₅equals hydrogen or C₁-C₆ alkyl as well as physiologically toleratedsalts and esters thereof.
 34. A peptide of 25-30 amino acids in lengthand having at least 25 contiguous amino acids derived from SEQ ID NO:1or SEQ ID NO:2 SEQ ID NO:1 1               5                   10 HisGly Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln    15                  20                  25 Met Glu Glu Glu Ala ValArg Leu Phe Ile Glu Trp Leu             30 Lys Asn Glu Xaa or SEQ IDNO:2 1               5                   10 His Gly Glu Gly Thr Phe ThrSer Asp Leu Ser Lys Gln     15                  20                  25Met Glu Glu Glu Ala Val Arg Leu Phe Ile Glu Trp Leu             30 LysAsn Glu Xaa

in which Xaa specifies one of the proteinogenic amino acids: Asp, Glu,Arg, His, Lys, Ala, Asn, Cys, Gln, Ile, Leu, Met, Phe, Pro, Ser, Thr,Trp, Tyr, Val or a non-proteinogenic amino acid, and wherein inpositions 1, 2, 28, 29 or 30, independent of one another, amino acidsare optionally part of the sequence, and the N-terminus is representedby NR₁R₂ in which R₁ denotes hydrogen, acetyl, trifluoroacetyl,adamantyl, Fmoc, benzyloxycarbonyl, Boc, Alloc, C₁-C₆ alkyl, C₂-C₈alkenyl or C₇-C₉ aralkyl, R₁ denotes hydrogen, acetyl, trifluoroacetyl,adamantyl, Fmoc, benzyloxycarbonyl, Boc, Alloc, C₁-C₆ alkyl, C₂-C₈alkenyl or C₇-C₉ aralkyl, and the C-terminus is represented by COR₃ inwhich R₃ equals OR₄ or NR₄R₅ in which R₄ equals hydrogen or C₁-C₆ alkylin which R₅ equals hydrogen or C₁-C₆ alkyl as well as physiologicallytolerated salts and esters thereof, with the proviso that at least onebut at most 10 of the following modifications (a) to (o) apply to theamino acid chain: (a) the α-amino acid in position 1 is D-His, Ala,D-Ala, Gly, Lys or D-Lys; (b) the α-amino acid in position 2 is Ser,D-Ser, Thr, D-Thr, Ala, D-Ala, Ile, D-Ile, Val, D-Val, Leu, D-Leu, Cys,Asp, Glu, Phe, His, Lys, Met, Asn, Pro, Gln, Arg, Trp or Tyr; (c) theα-amino acid in position 3 is D-Glu, D-Asp, Ala or D-Ala; (d) the aminoacid in position 4 is Ala, D-Ala or β-Ala; (e) the α-amino acid inposition 5 is Ser, Tyr or Ala; (f) the α-amino acid in position 6 isAla, Ile, Val, Leu, Cha or Tyr; (g) the α-amino acid in position 7 isAla, D-Ala, Tyr, D-Tyr, Ser, D-Ser or D-Thr; (h) the α-amino acid inposition 8 is Ala, Tyr or Thr; (i) the α-amino acid in position 9 isAla, D-Ala, Glu, D-Glu or D-Asp; (j) the amino acids in positions 10,11, 12, 15, 16, 17, 18, 19, 20, 21, 24, 28, 29 are, independent of oneanother, one of the above-specified proteinogenic amino acids which aredifferent from the amino acid already present in the respectiveposition(s) in SEQ ID NO:1 or SEQ ID NO:2 or a non-proteinogenic D- orL-amino acid; (k) the α-amino acid in position 13 is a neutral L-aminoacid except Gln: (l) the α-amino acid in position 14 is replaced by aneutral L- or D-amino acid, except Met or L-leucine; (m) the α-aminoacid in position 22 is D-Phe, Tyr, D-Tyr, Leu, D-Leu, Val, D-Val, L-Cha,D-Cha, β-1-Nal, β-2-Nal or β-1-D-Nal; (n) the α-amino acid in position23 is Leu, D-Leu, D-Ile, Val, D-Val, L-Cha, D-Cha, Tyr, D-Tyr, Phe orD-Phe; and (o) the α-amino acid in positions 25, 26 or 27 is a neutralL- or D-amino acid, except Trp for position 25, Leu for position 26 andLys for position
 27. 35. A peptide as claimed in claim 34, wherein inSEQ ID NO:1 or SEQ ID NO:2 an amino acid has been substituted atposition
 2. 36. A peptide as claimed in claim 34, wherein in SEQ ID NO:1or SEQ ID NO:2 an amino acid has been substituted at position
 14. 37. Apeptide as claimed in claim 34, wherein in SEQ ID NO:1 or SEQ ID NO:2 anamino acid has been substituted at position
 3. 38. A peptide as claimedin claim 34, wherein in SEQ ID NO:1 or SEQ ID NO:2 the α-amino acid inposition 1 is Ala, Gly or Lys.
 39. A peptide as claimed in claim 34,wherein in SEQ ID NO:1 or SEQ ID NO:2 the α-amino acid in position 2 isThr, Ala, Val, Ile or Leu.
 40. A peptide as claimed in claim 34, whereinin SEQ ID NO:1 or SEQ ID NO:2 the α-amino acid in position 3 is Ala. 41.A peptide as claimed in claim 34, wherein in SEQ ID NO:1 or NO.2 theα-amino acid in position 4 is Ala.
 42. A peptide as claimed in claim 34,wherein in SEQ ID NO:1 or SEQ ID NO:2 the α-amino acid in position 6 isAla, Ile, Val, Leu or Tyr.
 43. A peptide as claimed in claim 34, whereinin SEQ ID NO:1 or SEQ ID NO:2 the α-amino acid in position 7 is Ala, Tyror Ser.
 44. A peptide as claimed in claim 34, wherein in SEQ ID NO:1 orSEQ ID NO:2 the α-amino acid in position 9 is Ala or Glu.
 45. A peptideas claimed in claim 34, wherein in SEQ ID NO:1 or SEQ ID NO:2 theα-amino acid in positions 10, 11, 12, 15, 16, 17, 18, 19, 20, 21, 24,28, 29 is a proteinogenic L-amino acid.
 46. A peptide as claimed inclaim 34, wherein in SEQ ID NO:1 or SEQ ID NO:2 the α-amino acid inposition 13 is a neutral proteinogenic L-amino acid.
 47. A peptide asclaimed in claim 34, wherein in SEQ ID NO:1 or SEQ ID NO:2 the α-aminoacid in position 14 is Nle, D-Nle, Ala, D-Ala, Ile, D-Ile, Val or D-Val.48. A peptide as claimed in claim 34, wherein in SEQ ID NO:1 or SEQ IDNO:2 the α-amino acid in position 14 is Ile, Val or Ala.
 49. A peptideas claimed in claim 34, wherein in SEQ ID NO:1 or SEQ ID NO:2 theα-amino acid in position 22 is Tyr, Leu or Val.
 50. A peptide as claimedin claim 34, wherein in SEQ ID NO:1 or SEQ ID NO:2 the α-amino acid inposition 23 is Leu, Val, Tyr or Phe.
 51. A peptide as claimed in claim34, wherein in SEQ ID NO:1 or SEQ ID NO:2 the α-amino acid in positions25, 26 or 27 is a proteinogenic L-amino acid.
 52. A peptide as claimedin claim 34 wherein the α-amino acid in position 30 is Arg or Tyr.
 53. Amethod of stimulating the release of insulin in a mammal comprisingadministering to the mammal a peptide according to claim 28 in an amountsufficient to release insulin.
 54. The method of claim 53, wherein themammal is a human.